Salt forms and polymorphs of (r)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido) pyridazin-3-yl)-2-fluorobutyl)-n-methyl-1H-1,2,3-triazole-4-carboxamide

ABSTRACT

Disclosed herein is the compound (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido) pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide, and salt forms and polymorphs thereof demonstrating improved exposure after oral dosing. Methods of inhibition GLS1 activity in a human or animal subject are also provided.

This application is a continuation of U.S. application Ser. No.16/241,596, filed Jan. 7, 2019, which is a continuation of U.S.application Ser. No. 16/172,506, filed Oct. 26, 2018, which is adivisional of U.S. application Ser. No. 15/387,560, filed Dec. 21, 2016,which claims the benefit of priority of U.S. Provisional application No.62/271,018, filed Dec. 22, 2015, the disclosures of which are herebyincorporated by reference as if written herein in their entireties. Thisapplication also incorporates by reference the disclosure of U.S.application Ser. No. 14/791,186, filed Jul. 2, 2015, in its entirety.

The present disclosure relates to new heterocyclic compounds, salts andpolymorphs thereof, compositions, and their application aspharmaceuticals for the treatment of disease. Methods of inhibition ofGLS1 activity in a human or animal subject are also provided for thetreatment of diseases such as cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an overlay of XRPD of Example 1 after purification in varioussolvents.

FIG. 2 is an XRPD of Example 1 after recrystallization in acetone(Polymorph D).

FIG. 3 depicts polarized light microscopy of Example 1 afterrecrystallization in acetone (Polymorph D).

FIG. 4 depicts DSC and TGA behavior of Example 1 after purification inacetone (Polymorph D).

FIG. 5 displays the IR spectrum of purified free base Example 1(Polymorph D).

FIG. 6 displays the ¹H NMR spectrum of purified free base Example 1(Polymorph D).

FIG. 7 depicts DVS of purified free base Example 1 (Polymorph D). Thetop graphdepicts change in mass as a function of P/Po (the ordinate,“Change in Mass (%)—Ref”, goes from 0 to 0.2 in increments of 0.02; theabscissa, “Target % P/Po”, from 0 to 100 in increments of 10). Thebottom graph depicts change of mass as a function of time (the ordinate,“Change in Mass (%)—Ref”, goes from 0 to 0.2 in increments of 0.02; theabscissa, “Time/mins”, from 0 to 200 in increments of 20).

FIG. 8 is an overlay of the XRPD of Polymer D before and after DVSanalysis.

FIG. 9 is an overlay of the XRPD for salt candidates prepared by Method1.

FIG. 10 is an overlay of the XRPD for additional salt candidatesprepared by Method 1.

FIG. 11 is an overlay of the XRPD for the chloride salt asrecrystallized from various solvents.

FIG. 12 is an overlay of the XRPD for the sulfate salt as recrystallizedfrom various solvents.

FIG. 13 is an overlay of the XRPD for the methanesulfonate salt asrecrystallized from various solvents.

FIG. 14 is an overlay of the XRPD for salt candidates prepared by Method2.

FIG. 15 is an overlay of the XRPD for salt candidates prepared by Method3.

FIG. 16 is an overlay of the XRPD for the chloride salt as prepared byMethod 3 and recrystallized from EtOAc.

FIG. 17 is an overlay of the XRPD for the sulfate salt as prepared byMethod 3 and recrystallized from EtOAc.

FIG. 18 is an overlay of the XRPD for the mesylate salt as prepared byMethod 3 and recrystallized from EtOAc.

FIG. 19 is an overlay of the XRPD for the tosylate salt as prepared byMethod 3 and recrystallized from EtOAc.

FIG. 20 is an overlay of the XRPD for the salt candidates from theMethod 3 scale-up.

FIG. 21 is an XRPD of the mesylate salt as prepared by the Method 3scale-up.

FIG. 22 is an XRPD of the chloride salt as prepared by the Method 3scale-up and recrystallized from EtOAc.

FIG. 23 is an XRPD of the tosylate salt as prepared by the Method 3scale-up and recrystallized from EtOAc.

FIG. 24 depicts the ¹H NMR in MeOD of the scale-up of purified chloridesalt of Example 1.

FIG. 25 depicts the ¹H NMR in MeOD of the scale-up of purified mesylatesalt of Example 1.

FIG. 26 depicts the ¹H NMR in MeOD of the scale-up of purified tosylatesalt of Example 1.

FIG. 27 depicts the PLM of the scale-up of purified chloride salt ofExample 1.

FIG. 28 depicts the PLM of the scale-up of purified mesylate salt ofExample 1.

FIG. 29 depicts the PLM of the scale-up of purified tosylate salt ofExample 1.

FIG. 30 depicts an overlay of the DSC and TGA of the scale-up of thepurified chloride salt of Example 1.

FIG. 31 depicts an overlay of the DSC and TGA of the scale-up of thepurified mesylate salt of Example 1.

FIG. 32 depicts an overlay of the DSC and TGA of the scale-up of thepurified tosylate salt of Example 1.

Metabolic deregulation is a hallmark of cancer as tumors exhibit anincreased demand for nutrients and macromolecules to fuel their rapidproliferation. Glutamine (Gln), the most abundant amino acid incirculation, plays an essential role in providing cancer cells withbiosynthetic intermediates required to support proliferation andsurvival. Specifically, glutaminolysis, or the enzymatic conversion ofglutamine to glutamate, provides proliferating cancer cells with asource of nitrogen for amino acid and nucleotide synthesis, and a carbonskeleton to fuel ATP and NADPH synthesis through the TCA cycle. Inaddition to supporting cell growth, glutamine metabolism plays acritical role in maintaining cellular redox homeostasis as glutamate canbe converted into glutathione, the major intracellular antioxidant.

Glutaminolysis is regulated by mitochondrial glutaminase (GLS), the ratelimiting enzyme that catalyzes the conversion of Gln to glutamate andammonia. Mammalian cells contain 2 genes that encode glutaminase: thekidney-type (GLS1) and liver-type (GLS2) enzymes. Each has been detectedin multiple tissue types, with GLS1 being widely distributed throughoutthe body. GLS1 is a phosphate-activated enzyme that exists in humans astwo major splice variants, a long form (referred to as KGA) and a shortform (GAC), which differ only in their C-terminal sequences. Both formsof GLS1 are thought to bind to the inner membrane of the mitochondrionin mammalian cells, although at least one report suggests thatglutaminase may exist in the intramembrane space, dissociated from themembrane. GLS is frequently overexpressed in human tumors and has beenshown to be positively regulated by oncogenes such as Myc. Consistentwith the observed dependence of cancer cell lines on glutaminemetabolism, pharmcological inhibition of GLS offers the potential totarget Gln addicted tumors.

Thus, there is a need for glutaminase inhibitors that are specific andcapable of being formulated for in vivo use.

Accordingly, disclosed herein are new compositions and methods forinhibiting glutaminase activity.

Provided is the compound

or a salt, solvate, or polymorph thereof.

Also provided is the compound

or a salt, solvate, or polymorph thereof.

Also provided is the compound

or a salt, solvate, or polymorph thereof.

In certain embodiments, the compound is a solvate. In certainembodiments, the solvate is DMSO. In certain embodiments, wherein theDMSO is associated with the compound in a 1:1 ratio.

Also provided is a salt of structural Formula I

or a solvate or polymorph thereof, wherein:

R⁻ is chosen from Cl⁻, Br⁻, I⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻, CH₃SO₃ ⁻, PhSO₃⁻, 4-MePhSO₃ ⁻, and NaphthaleneSO₃ ⁻;

n is an integer from 0 to 2; and

Y is an optional solvate.

In certain embodiments, R⁻ is chosen from Cl⁻, HSO₄ ⁻, CH₃SO₃ ⁻, and4-MePhSO₃ ⁻.

In certain embodiments, n is 1.

In certain embodiments, Y is absent (i.e., the salt is anhydrous).

In certain embodiments, R⁻ is CH₃SO₃ ⁻ (i.e., the salt is the mesylate).

In certain embodiments, the mesylate salt is characterized as having oneor more x-ray powder diffraction peaks chosen from about 9.2, about10.8, about 13.8, about 16.7, about 17.3, about 18.4, about 18.7, about19.9, about 20.6, about 21.4, about 22.1, about 22.3, about 22.6, about22.9, about 24.1, and about 32.1 degrees 2-theta. In certainembodiments, the salt has three or more of the peaks. In certainembodiments, the salt has five or more of the peaks.

In certain embodiments, the mesylate salt displays an endothermic peakin DSC with onset of 180° C.±1° C. In certain embodiments, the mesylatesalt displays weight loss in DSC of less than 1.0% from 30° C. to 200°C. In certain embodiments, the mesylate salt is anhydrous.

In certain embodiments, R⁻ is Cl⁻ (i.e., the salt is the chloride).

In certain embodiments, the chloride salt is characterized as having oneor more x-ray powder diffraction peaks chosen from about 4.6, about9.26, about 11.0, about 12.6, about 13.2, about 13.8, about 16.5, about19.0, about 20.8, about 22.0, about 22.4, about 22.7, about 24.2, about25.0, and about 33.4 degrees 2-theta. In certain embodiments, the salthas three or more of the peaks. In certain embodiments, the salt hasfive or more of the peaks.

In certain embodiments, R⁻ is 4-MePhSO₃ ⁻ (i.e., the salt is thetosylate).

In certain embodiments, the tosylate salt is characterized as having oneor more x-ray powder diffraction peaks chosen from about 4.5, about 9.0,about 10.3, about 10.5, about 10.7, about 11.1, about 11.7, about 13.6,about 14.3, about 17.1, about 17.3, about 17.6, about 18.5, about 18.9,about 19.0, about 19.2, about 19.8, about 20.1, about 20.4, about 20.8,about 21.4, about 21.8, about 22.4, about 22.6, about 23.4, about 24.3,about 25.1, about 26.0, about 26.3, about 27.2, about 27.4, and about28.2 degrees 2-theta. In certain embodiments, the salt has three or moreof the peaks. In certain embodiments, the salt has five or more of thepeaks.

In certain embodiments, the tosylate salt displays an endothermic peakin DSC with onset of 185° C.±1° C. In certain embodiments, the tosylatesalt displays weight loss in DSC of less than 1.0% from 30° C. to 200°C. In certain embodiments, the tosylate salt is anhydrous.

In certain embodiments, R⁻ is HSO₄ ⁻ (i.e., the salt is the sulfatesalt).

Also provided is the solid compound

or a polymorph thereof. In certain embodiments, the compound orpolymorph thereof is crystalline.

In certain embodiments, the polymorph is Polymorph A.

In certain embodiments, the polymorph is Polymorph B.

In certain embodiments, the polymorph is Polymorph C.

In certain embodiments, the polymorph is Polymorph D.

Solid Polymorph D of the compound

is provided herein.

In certain embodiments, Polymorph D is characterized as having one ormore x-ray powder diffraction peaks chosen from about 4.0, about 8.0,about 11.6, about 11.9, about 14.9, about 15.9, about 17.6, about 19.9,about 20.2, about 22.4, about 23.7, and about 23.9 degrees 2-theta. Incertain embodiments, Polymorph D has three or more of the peaks. Incertain embodiments, Polymorph D has five or more of the peaks.

In certain embodiments, Polymorph D displays an endothermic peak in DSCwith onset of 197° C.±1° C. In certain embodiments, Polymorph D isanhydrous. In certain embodiments, Polymorph D displays weight loss inDSC of less than 1% from 30° C. to 200° C.

Also provided is a composition comprising a compound, salt, solvate, orpolymorph as recited herein, and a pharmaceutically acceptable carrier,adjuvant, or vehicle.

Also provided is a method of inhibiting GLS1 activity in a biologicalsample comprising contacting the biological sample with a compound,salt, solvate, or polymorph as recited herein.

Also provided is a A method of treating a GLS1-mediated disorder in asubject in need thereof, comprising the step of administering to thesubject a compound, salt, solvate, or polymorph as recited herein.

In certain embodiments, the subject is a human.

In certain embodiments, the GLS1-mediated disorder is chosen fromcancer, immunological disorders, and neurological disorders.

In certain embodiments, the GLS1-mediated disorder is cancer.

In certain embodiments, the cancer is chosen from Acute LymphoblasticLeukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma,AIDS-Related Cancers (Kaposi Sarcoma and Lymphoma), Anal Cancer,Appendix Cancer, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma,Bile Duct Cancer (including Extrahepatic), Bladder Cancer, Bone Cancer(including Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumor(such as Astrocytomas, Brain and Spinal Cord Tumors, Brain Stem Glioma,Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Central NervousSystem Embryonal Tumors, Craniopharyngioma, Ependymoblastoma,Ependymoma, Medulloblastoma, Medulloepithelioma, Pineal ParenchymalTumors of Intermediate Differentiation, Supratentorial PrimitiveNeuroectodermal Tumors and Pineoblastoma), Breast Cancer, BronchialTumors, Burkitt Lymphoma, Basal Cell Carcinoma, Bile Duct Cancer(including Extrahepatic), Bladder Cancer, Bone Cancer (includingOsteosarcoma and Malignant Fibrous Histiocytoma), Carcinoid Tumor,Carcinoma of Unknown Primary, Central Nervous System (such as AtypicalTeratoid/Rhabdoid Tumor, Embryonal Tumors and Lymphoma), CervicalCancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL),Chronic Myelogenous Leukemia (CML), Chronic MyeloproliferativeDisorders, Colon Cancer, Colorectal Cancer, Craniopharyngioma, CutaneousT-Cell Lymphoma (Mycosis Fungoides and Sézary Syndrome), Duct, Bile(Extrahepatic), Ductal Carcinoma In Situ (DCIS), Embryonal Tumors(Central Nervous System), Endometrial Cancer, Ependymoblastoma,Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing SarcomaFamily of Tumors, Extracranial Germ Cell Tumor, Extragonadal Germ CellTumor, Extrahepatic Bile Duct Cancer, Eye Cancer (like IntraocularMelanoma, Retinoblastoma), Fibrous Histiocytoma of Bone (includingMalignant and Osteosarcoma) Gallbladder Cancer, Gastric (Stomach)Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal StromalTumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal, Ovarian),Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head andNeck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis,Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer, IntraocularMelanoma, Islet Cell Tumors (Endocrine, Pancreas), Kaposi Sarcoma,Kidney (including Renal Cell), Langerhans Cell Histiocytosis, LaryngealCancer, Leukemia (including Acute Lymphoblastic (ALL), Acute Myeloid(AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), HairyCell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSézary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer (such as Epithelial, GermCell Tumor, and Low Malignant Potential Tumor), Pancreatic Cancer(including Islet Cell Tumors), Papillomatosis, Paraganglioma, ParanasalSinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, PrimaryCentral Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer,Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional CellCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma(like Ewing Sarcoma Family of Tumors, Kaposi, Soft Tissue, Uterine),Sézary Syndrome, Skin Cancer (such as Melanoma, Merkel Cell Carcinoma,Nonmelanoma), Small Cell Lung Cancer, Small Intestine Cancer, SoftTissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer withOccult Primary, Metastatic, Stomach (Gastric) Cancer, SupratentorialPrimitive Neuroectodermal Tumors, T-Cell Lymphoma (Cutaneous, MycosisFungoides and Sézary Syndrome), Testicular Cancer, Throat Cancer,Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancerof the Renal Pelvis and Ureter, Trophoblastic Tumor (Gestational),Unknown Primary, Unusual Cancers of Childhood, Ureter and Renal Pelvis,Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial,Uterine Sarcoma, Waldenström Macroglobulinemia and Wilms Tumor, or avariant thereof.

Also provided herein is a method of treating a GLS1-mediated disorder ina subject in need thereof, comprising the sequential orco-administration of a compound, salt, solvate, or polymorph as recitedherein, and another therapeutic agent.

In certain embodiments, the therapeutic agent is chosen from a taxane,inhibitor of bcr-abl, inhibitor of EGFR, DNA damaging agent, andantimetabolite.

In certain embodiments, the therapeutic agent is chosen fromaminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine,carboplatin, carmustine, chlorambucil, chloroquine, cisplatin,cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin,dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane,filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone,flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole,leucovorin, leuprolide, levamisole, lomustine, lonidamine,mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, perifosine, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, sorafenib, streptozocin,sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide,testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan,trastuzumab, tretinoin, vinblastine, vincristine, vindesine, andvinorelbine.

In certain embodiments, the therapeutic agent is docetaxel.

In certain embodiments, the method further comprises administeringnon-chemical methods of cancer treatment.

In certain embodiments, the method further comprises administeringradiation therapy.

In certain embodiments, the method further comprises administeringsurgery, thermoablation, focused ultrasound therapy, cryotherapy, or anycombination thereof.

Also provided herein is a compound, salt, solvate, or polymorph asrecited herein for use in human therapy.

Also provided herein is a compound, salt, solvate, or polymorph asrecited herein for use in treating a GLS1-mediated disease.

Also provided herein is the use of a compound, salt, solvate, orpolymorph as recited herein for the manufacture of a medicament to treata GLS1-mediated disease.

DETAILED DESCRIPTION

Abbreviations and Definitions

To facilitate understanding of the disclosure, a number of terms andabbreviations as used herein are defined below as follows:

When introducing elements of the present disclosure or the preferredembodiment(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B, i.e. A alone, Balone or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination or A, B, and C incombination.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n₂ are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues that it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the disclosure encompasses all stereochemicalisomeric forms, including diastereomeric, enantiomeric, and epimericforms, as well as d-isomers and 1-isomers, and mixtures thereof.Individual stereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentdisclosure includes all cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers as well as the appropriate mixtures thereof.Additionally, compounds may exist as tautomers; all tautomeric isomersare provided by this disclosure. Additionally, the compounds disclosedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. In general, the solvated forms are considered equivalent to theunsolvated forms.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

GLS1 inhibitor is used herein to refer to a compound that exhibits anIC50 with respect to GLS1 activity of no more than about 100 μM and moretypically not more than about 50 μM, as measured in the GLS1 enzymeassay described generally herein below. IC50 is that concentration ofinhibitor that reduces the activity of an enzyme (e.g., GLS1) tohalf-maximal level. Certain compounds disclosed herein have beendiscovered to exhibit inhibition against GLS1. In certain embodiments,compounds will exhibit an IC50 with respect to GLS1 of no more thanabout 10 μM; in further embodiments, compounds will exhibit an IC50 withrespect to GLS1 of no more than about 5 μM; in yet further embodiments,compounds will exhibit an IC50 with respect to GLS1 of not more thanabout 1 μM; in yet further embodiments, compounds will exhibit an IC50with respect to GLS1 of not more than about 200 nM, as measured in theGLS1 binding assay described herein.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. Treatment may also be preemptive in nature, i.e.,it may include prevention of disease. Prevention of a disease mayinvolve complete protection from disease, for example as in the case ofprevention of infection with a pathogen, or may involve prevention ofdisease progression. For example, prevention of a disease may not meancomplete foreclosure of any effect related to the diseases at any level,but instead may mean prevention of the symptoms of a disease to aclinically significant or detectable level. Prevention of diseases mayalso mean prevention of progression of a disease to a later stage of thedisease.

The term “patient” is generally synonymous with the term “subject” andincludes all mammals including humans. Examples of patients includehumans, livestock (farm animals) such as cows, goats, sheep, pigs, andrabbits, and companion animals such as dogs, cats, rabbits, and horses.Preferably, the patient is a human.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present disclosure includes compounds listed above in theform of salts, including acid addition salts. Suitable salts includethose formed with both organic and inorganic acids. Such acid additionsalts will normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate(HSO₄ ⁻), butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride (HCl,chloride, Cl⁻), hydrobromide (HBr, bromide, Br⁻), hydroiodide (HI,iodide, I⁻), 2-hydroxyethansulfonate (isethionate), lactate, maleate,malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate (mesylate,MsOH, MeSO₃H, CH₃SO₃H, CH₃SO₃ ⁻), naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, nitrate (NO₃ ⁻), oxalate, pamoate, pectinate,persulfate, 3-phenylproprionate, phosphonate, picrate, pivalate,propionate, pyroglutamate, succinate, sulfate (SO₄ ²⁻), sulfonate,tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate,glutamate, bicarbonate, para-toluenesulfonate (tosylate, TsOH, Ts,p-tosylate, methylbenzenesulfonate, 4-MePhSO₃ ⁻), phenylsulfonate (PhSO₃⁻), HOSO₂CH₂CH₂SO₃ ⁻, ⁻OSO₂CH₂CH₂SO₃ ⁻, and undecanoate. Acid additionsalts can be named in terms of the acid used to form the salt, or theanion present in the salt. Thus, for example, the terms “chloride salt”and “hydrochloride salt” are understood to represent the same salt.Also, basic groups in the compounds disclosed herein can be quaternizedwith methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and steryl chlorides, bromides, and iodides; and benzyl andphenethyl bromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric. Salts can also beformed by coordination of the compounds with an alkali metal or alkalineearth ion. Hence, the present disclosure contemplates sodium, potassium,magnesium, and calcium salts of the compounds disclosed herein, and thelike.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid.

Compounds

The present disclosure provides the compound

or a salt, solvate, or polymorph thereof.

Also provided is the compound

or a salt, solvate, or polymorph thereof.

Also provided is the compound

or a salt, solvate, or polymorph thereof.

Also provided is a salt of structural Formula I, or an embodimentthereof, as disclosed herein.

In certain embodiments, the compound, salt, or polymorph thereof ischosen from:

Pharmaceutical Compositions

While it may be possible for the compounds of the subject disclosure tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein may be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject disclosure or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Compounds described herein can be administered as follows:

Oral Administration

The compounds of the present invention may be administered orally,including swallowing, so the compound enters the gastrointestinal tract,or is absorbed into the blood stream directly from the mouth, includingsublingual or buccal administration.

Suitable compositions for oral administration include solid formulationssuch as tablets, pills, cachets, lozenges and hard or soft capsules,which can contain liquids, gels, powders, or granules.

In a tablet or capsule dosage form the amount of drug present may befrom about 0.05% to about 95% by weight, more typically from about 2% toabout 50% by weight of the dosage form.

In addition, tablets or capsules may contain a disintegrant, comprisingfrom about 0.5% to about 35% by weight, more typically from about 2% toabout 25% of the dosage form. Examples of disintegrants include methylcellulose, sodium or calcium carboxymethyl cellulose, croscarmellosesodium, polyvinylpyrrolidone, hydroxypropyl cellulose, starch and thelike.

Suitable binders, for use in a tablet, include gelatin, polyethyleneglycol, sugars, gums, starch, hydroxypropyl cellulose and the like.Suitable diluents, for use in a tablet, include mannitol, xylitol,lactose, dextrose, sucrose, sorbitol and starch.

Suitable surface active agents and glidants, for use in a tablet orcapsule, may be present in amounts from about 0.1% to about 3% byweight, and include polysorbate 80, sodium dodecyl sulfate, talc andsilicon dioxide.

Suitable lubricants, for use in a tablet or capsule, may be present inamounts from about 0.1% to about 5% by weight, and include calcium, zincor magnesium stearate, sodium stearyl fumarate and the like.

Tablets may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed withbinders, inert diluents, or lubricating, surface active or dispersingagents. Molded tablets may be made by molding in a suitable machine amixture of the powdered compound moistened with a liquid diluent. Dyesor pigments may be added to tablets for identification or tocharacterize different combinations of active compound doses.

Liquid formulations can include emulsions, solutions, syrups, elixirsand suspensions, which can be used in soft or hard capsules. Suchformulations may include a pharmaceutically acceptable carrier, forexample, water, ethanol, polyethylene glycol, cellulose, or an oil. Theformulation may also include one or more emulsifying agents and/orsuspending agents.

Compositions for oral administration may be formulated as immediate ormodified release, including delayed or sustained release, optionallywith enteric coating.

In another embodiment, a pharmaceutical composition comprises atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Parenteral Administration

Compounds of the present invention may be administered directly into theblood stream, muscle, or internal organs by injection, e.g., by bolusinjection or continuous infusion. Suitable means for parenteraladministration include intravenous, intra-muscular, subcutaneousintraarterial, intraperitoneal, intrathecal, intracranial, and the like.Suitable devices for parenteral administration include injectors(including needle and needle-free injectors) and infusion methods. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials.

Most parenteral formulations are aqueous solutions containingexcipients, including salts, buffering, suspending, stabilizing and/ordispersing agents, antioxidants, bacteriostats, preservatives, andsolutes which render the formulation isotonic with the blood of theintended recipient, and carbohydrates.

Parenteral formulations may also be prepared in a dehydrated form (e.g.,by lyophilization) or as sterile non-aqueous solutions. Theseformulations can be used with a suitable vehicle, such as sterile water.Solubility-enhancing agents may also be used in preparation ofparenteral solutions.

Compositions for parenteral administration may be formulated asimmediate or modified release, including delayed or sustained release.Compounds may also be formulated as depot preparations. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Topical Administration

Compounds of the present invention may be administered topically (forexample to the skin, mucous membranes, ear, nose, or eye) ortransdermally. Formulations for topical administration can include, butare not limited to, lotions, solutions, creams, gels, hydrogels,ointments, foams, implants, patches and the like. Carriers that arepharmaceutically acceptable for topical administration formulations caninclude water, alcohol, mineral oil, glycerin, polyethylene glycol andthe like. Topical administration can also be performed by, for example,electroporation, iontophoresis, phonophoresis and the like.

Typically, the active ingredient for topical administration may comprisefrom 0.001% to 10% w/w (by weight) of the formulation. In certainembodiments, the active ingredient may comprise as much as 10% w/w; lessthan 5% w/w; from 2% w/w to 5% w/w; or from 0.1% to 1% w/w of theformulation.

Compositions for topical administration may be formulated as immediateor modified release, including delayed or sustained release.

Rectal, Buccal, and Sublingual Administration

Suppositories for rectal administration of the compounds of the presentinvention can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di-, ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the drug.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

Administration by Inhalation

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray or powder. Pressurizedpacks may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Alternatively, foradministration by inhalation or insufflation, the compounds according tothe disclosure may take the form of a dry powder composition, forexample a powder mix of the compound and a suitable powder base such aslactose or starch. The powder composition may be presented in unitdosage form, in for example, capsules, cartridges, gelatin or blisterpacks from which the powder may be administered with the aid of aninhalator or insufflator.

Other carrier materials and modes of administration known in thepharmaceutical art may also be used. Pharmaceutical compositions of theinvention may be prepared by any of the well-known techniques ofpharmacy, such as effective formulation and administration procedures.Preferred unit dosage formulations are those containing an effectivedose, as herein recited, or an appropriate fraction thereof, of theactive ingredient. The precise amount of compound administered to apatient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. In addition, the route ofadministration may vary depending on the condition and its severity. Theabove considerations concerning effective formulations andadministration procedures are well known in the art and are described instandard textbooks. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds.,Handbook of Pharmaceutical Excipients (3^(rd) Ed.), AmericanPharmaceutical Association, Washington, 1999.

Methods of Treatment

The present disclosure provides compounds and pharmaceuticalcompositions that inhibit glutaminase activity, particularly GLS1activity and are thus useful in the treatment or prevention of disordersassociated with GLS1. Compounds and pharmaceutical compositions of thepresent disclosure selectively modulate GLS1 and are thus useful in thetreatment or prevention of a range of disorders associated with GLS1 andinclude, but are not limited to, cancer, immunological or neurologicaldiseases associated with GLS1.

Neurological Disorders

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment or prevention ofneurological diseases.

The most common neurotransmitter is glutamate, derived from theenzymatic conversion of glutamine via glutaminase. High levels ofglutamate have been shown to be neurotoxic. Following traumatic insultto neuronal cells, there occurs a rise in neurotransmitter release,particularly glutamate. Accordingly, inhibition of glutaminase has beenhypothesized as a means of treatment following an ischemic insult, suchas stroke.

Huntington's disease is a progressive, fatal neurological condition. Ingenetic mouse models of Huntington's disease, it was observed that theearly manifestation of the disease correlated with dysregulatedglutamate release (Raymond et al., Neuroscience, 2011). InHIV-associated dementia, HIV infected macrophages exhibit upregulatedglutaminase activity and increased glutamate release, leading toneuronal damage (Huang et al., J. Neurosci., 2011). Similarly, inanother neurological disease, the activated microglia in Rett Syndromerelease glutamate causing neuronal damage. The release of excessglutamate has been associated with the up-regulation of glutaminase(Maezawa et al., J. Neurosci, 2010). In mice bred to have reducedglutaminase levels, sensitivity to psychotic-stimulating drugs, such asamphetamines, was dramatically reduced, thus suggesting that glutaminaseinhibition may be beneficial in the treatment of schizophrenia(Gaisler-Salomon et al., Neuropsychopharmacology, 2009). Bipolardisorder is a devastating illness that is marked by recurrent episodesof mania and depression. This disease is treated with mood stabilizerssuch as lithium and valproate; however, chronic use of these drugsappear to increase the abundance of glutamate receptors (Nanavati etal., J. Neurochem., 2011), which may lead to a decrease in the drug'seffectiveness over time. Thus, an alternative treatment may be to reducethe amount of glutamate by inhibiting glutaminase. This may or may notbe in conjunction with the mood stabilizers. Memantine, a partialantagonist of N-methyl-D-aspartate receptor (NMDAR), is an approvedtherapeutic in the treatment of Alzheimer's disease. Currently, researchis being conducted looking at memantine as a means of treating vasculardementia and Parkinson's disease (Oliverares et al., Curr. AlzheimerRes., 2011). Since memantine has been shown to partially block the NMDAglutamate receptor also, it is not unresasonable to speculate thatdecreasing glutamate levels by inhibiting glutaminase could also treatAlzheimer's disease, vascular dementia and Parkinson's disease.Alzheimer's disease, bipolar disorder, HIV-associated dementia,Huntington's disease, ischemic insult, Parkinson's disease,schizophrenia, stroke, traumatic insult and vascular dementia are but afew of the neurological diseases that have been correlated to increasedlevels of glutamate. Thus, inhibiting glutaminase with a compounddescribed herein can reduce or prevent neurological diseases. Therefore,in certain embodiments, the compounds may be used for the treatment orprevention of neurological diseases.

Immunological Disorders

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment or prevention ofimmunological diseases.

Activation of T lymphocytes induces cell growth, proliferation, andcytokine production, thereby placing energetic and biosynthetic demandson the cell. Glutamine serves as an amine group donor for nucleotidesynthesis, and glutamate, the first component in glutamine metabolism,plays a direct role in amino acid and glutathione synthesis, as well asbeing able to enter the Krebs cycle for energy production (Carr et al.,J. Immunol., 2010). Mitogen-induced T cell proliferation and cytokineproduction require high levels of glutamine metabolism, thus inhibitingglutaminase may serve as a means of immune modulation. In multiplesclerosis, an inflammatory autoimmune disease, the activated microgliaexhibit up-regulated glutaminase and release increased levels ofextracellular glutamate. Glutamine levels are lowered by sepsis, injury,burns, surgery and endurance exercise (Calder et al., Amino Acids,1999). These situations put the individual at risk of immunosuppression.In fact, in general, glutaminase gene expression and enzyme activity areboth increased during T cell activity. Patients given glutaminefollowing bone marrow transplantation resulted in a lower level ofinfection and reduced graft v. host disease (Crowther, Proc. Nutr. Soc.,2009). T cell proliferation and activiation is involved in manyimmunological diseases, such as inflammatory bowel disease, Crohn'sdisease, sepsis, psoriasis, arthritis (including rheumatoid arthritis),multiple sclerosis, graft v. host disease, infections, lupus anddiabetes. In an embodiment of the invention, the compounds describedherein can be used to treat or prevent immunological diseases.

Cancer

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment or prevention ofcancer.

In addition to serving as the basic building blocks of proteinsynthesis, amino acids have been shown to contribute to many processescritical for growing and dividing cells, and this is particularly truefor cancer cells. Nearly all definitions of cancer include reference todysregulated proliferation. Numerous studies on glutamine metabolism incancer indicate that many tumors are avid glutamine consumers (Souba,Ann. Surg., 1993; Collins et al., J. Cell. Physiol., 1998; Medina, J.Nutr., 2001; Shanware et al., J. Mol. Med., 2011). An embodiment of theinvention is the use of the compounds described herein for the treatmentof cancer.

In some embodiments, the compounds of the present disclosure may be usedto prevent or treat cancer, wherein the cancer is one or a variant ofAcute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma andLymphoma), Anal Cancer, Appendix Cancer, Atypical Teratoid/RhabdoidTumor, Basal Cell Carcinoma, Bile Duct Cancer (including Extrahepatic),Bladder Cancer, Bone Cancer (including Osteosarcoma and MalignantFibrous Histiocytoma), Brain Tumor (such as Astrocytomas, Brain andSpinal Cord Tumors, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors,Craniopharyngioma, Ependymoblastoma, Ependymoma, Medulloblastoma,Medulloepithelioma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, BasalCell Carcinoma, Bile Duct Cancer (including Extrahepatic), BladderCancer, Bone Cancer (including Osteosarcoma and Malignant FibrousHistiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary, CentralNervous System (such as Atypical Teratoid/Rhabdoid Tumor, EmbryonalTumors and Lymphoma), Cervical Cancer, Childhood Cancers, Chordoma,Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma (Mycosis Fungoides andSézary Syndrome), Duct, Bile (Extrahepatic), Ductal Carcinoma In Situ(DCIS), Embryonal Tumors (Central Nervous System), Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer(like Intraocular Melanoma, Retinoblastoma), Fibrous Histiocytoma ofBone (including Malignant and Osteosarcoma) Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalStromal Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia,Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors (Endocrine, Pancreas), KaposiSarcoma, Kidney (including Renal Cell), Langerhans Cell Histiocytosis,Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), AcuteMyeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML),Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSézary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer (such as Epithelial, GermCell Tumor, and Low Malignant Potential Tumor), Pancreatic Cancer(including Islet Cell Tumors), Papillomatosis, Paraganglioma, ParanasalSinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, PrimaryCentral Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer,Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional CellCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma(like Ewing Sarcoma Family of Tumors, Kaposi, Soft Tissue, Uterine),Sézary Syndrome, Skin Cancer (such as Melanoma, Merkel Cell Carcinoma,Nonmelanoma), Small Cell Lung Cancer, Small Intestine Cancer, SoftTissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer withOccult Primary, Metastatic, Stomach (Gastric) Cancer, SupratentorialPrimitive Neuroectodermal Tumors, T-Cell Lymphoma (Cutaneous, MycosisFungoides and Sézary Syndrome), Testicular Cancer, Throat Cancer,Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancerof the Renal Pelvis and Ureter, Trophoblastic Tumor (Gestational),Unknown Primary, Unusual Cancers of Childhood, Ureter and Renal Pelvis,Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial,Uterine Sarcoma, Waldenström Macroglobulinemia or Wilms Tumor.

In certain embodiments, the cancer to be treated is one specific toT-cells such as T-cell lymphomia and lymphblastic T-cell leukemia.

In some embodiments, methods described herein are used to treat adisease condition comprising administering to a subject in need thereofa therapeutically effective amount of a compound of Formula I orpharmaceutically acceptable salt thereof, wherein the condition iscancer which has developed resistance to chemotherapeutic drugs and/orionizing radiation.

Combinations and Combination Therapy

The compounds of the present invention can be used, alone or incombination with other pharmaceutically active compounds, to treatconditions such as those previously described hereinabove. Thecompound(s) of the present invention and other pharmaceutically activecompound(s) can be administered simultaneously (either in the samedosage form or in separate dosage forms) or sequentially. Accordingly,in one embodiment, the present invention comprises methods for treatinga condition by administering to the subject a therapeutically-effectiveamount of one or more compounds of the present invention and one or moreadditional pharmaceutically active compounds.

In another embodiment, there is provided a pharmaceutical compositioncomprising one or more compounds of the present invention, one or moreadditional pharmaceutically active compounds, and a pharmaceuticallyacceptable carrier.

In another embodiment, the one or more additional pharmaceuticallyactive compounds is selected from the group consisting of anti-cancerdrugs, anti-proliferative drugs, and anti-inflammatory drugs.

GLS1 inhibitor compositions described herein are also optionally used incombination with other therapeutic reagents that are selected for theirtherapeutic value for the condition to be treated. In general, thecompounds described herein and, in embodiments where combination therapyis employed, other agents do not have to be administered in the samepharmaceutical composition and, because of different physical andchemical characteristics, are optionally administered by differentroutes. The initial administration is generally made according toestablished protocols and then, based upon the observed effects, thedosage, modes of administration and times of administration subsequentlymodified. In certain instances, it is appropriate to administer a GLS1inhibitor compound, as described herein, in combination with anothertherapeutic agent. By way of example only, the therapeutic effectivenessof a GLS1 inhibitor is enhanced by administration of another therapeuticagent (which also includes a therapeutic regimen) that also hastherapeutic benefit. Regardless of the disease, disorder or conditionbeing treated, the overall benefit experienced by the patient is eithersimply additive of the two therapeutic agents or the patient experiencesan enhanced (i.e., synergistic) benefit. Alternatively, if a compounddisclosed herein has a side effect, it may be appropriate to administeran agent to reduce the side effect; or the therapeutic effectiveness ofa compound described herein may be enhanced by administration of anadjuvant.

Therapeutically effective dosages vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically effective dosages of drugs and other agents for use incombination treatment regimens are documented methodologies. Combinationtreatment further includes periodic treatments that start and stop atvarious times to assist with the clinical management of the patient. Inany case, the multiple therapeutic agents (one of which is a GLS1inhibitor as described herein) may be administered in any order, orsimultaneously. If simultaneously, the multiple therapeutic agents areoptionally provided in a single, unified form, or in multiple forms (byway of example only, either as a single pill or as two separate pills).

In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses optionally varies from more than zeroweeks to less than twelve weeks.

In addition, the combination methods, compositions and formulations arenot to be limited to the use of only two agents, the use of multipletherapeutic combinations are also envisioned. It is understood that thedosage regimen to treat, prevent, or ameliorate the condition(s) forwhich relief is sought, is optionally modified in accordance with avariety of factors. These factors include the disorder from which thesubject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, the dosage regimen actually employedvaries widely, in some embodiments, and therefore deviates from thedosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein are optionally a combined dosage form or in separatedosage forms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy areoptionally also administered sequentially, with either agent beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen optionally calls for sequentialadministration of the active agents or spaced-apart administration ofthe separate active agents. The time between the multiple administrationsteps ranges from a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent.

In another embodiment, a GLS1 inhibitor is optionally used incombination with procedures that provide additional benefit to thepatient. A GLS1 inhibitor and any additional therapies are optionallyadministered before, during or after the occurrence of a disease orcondition, and the timing of administering the composition containing aGLS1 inhibitor varies in some embodiments. Thus, for example, a GLS1inhibitor is used as a prophylactic and is administered continuously tosubjects with a propensity to develop conditions or diseases in order toprevent the occurrence of the disease or condition. A GLS1 inhibitor andcompositions are optionally administered to a subject during or as soonas possible after the onset of the symptoms. While embodiments of thepresent invention have been shown and described herein, it will beobvious to those skilled in the art that such embodiments are providedby way of example only. Numerous variations, changes, and substitutionswill now occur to those skilled in the art without departing from theinvention. It should be understood that in some embodiments of theinvention various alternatives to the embodiments described herein areemployed in practicing the invention.

A GLS1 inhibitor can be used in combination with anti-cancer drugs,including but not limited to the following classes: alkylating agents,anti-metabolites, plant alkaloids and terpenoids, topoisomeraseinhibitors, cytotoxic antibiotics, angiogenesis inhibitors and tyrosinekinase inhibitors.

For use in cancer and neoplastic diseases a GLS1 inhibitor may beoptimally used together with one or more of the following non-limitingexamples of anti-cancer agents:

-   1) alkylating agents, including but not limited to carmustine,    chlorambucil (LEUKERAN), cisplatin (PLATIN), carboplatin    (PARAPLATIN), oxaliplatin (ELOXATIN), streptozocin (ZANOSAR),    busulfan (MYLERAN), dacarbazine, ifosfamide, lomustine (CCNU),    melphalan (ALKERAN), procarbazine (MATULAN), temozolomide (TEMODAR),    thiotepa, and cyclophosphamide (ENDOXAN);-   2) anti-metabolites, including but not limited to cladribine    (LEUSTATIN), mercaptopurine (PURINETHOL), thioguanine, pentostatin    (NIPENT), cytosine arabinoside (cytarabine, ARA-C), gemcitabine    (GEMZAR), fluorouracil (5-FU, CARAC), capecitabine (XELODA),    leucovorin (FUSILEV), methotrexate (RHEUMATREX), raltitrexed;-   3) antimitotics, which are often plant alkaloids and terpenoids, or    derivateves thereof, including but not limited to taxanes such as    docetaxel (TAXITERE) and paclitaxel (ABRAXANE, TAXOL); vinca    alkaloids such as vincristine (ONCOVIN), vinblastine, vindesine, and    vinorelbine (NAVELBINE);-   4) topoisomerase inhibitors, including but not limited to    camptothecin (CTP), irinotecan (CAMPTOSAR), topotecan (HYCAMTIN),    teniposide (VUMON), and etoposide (EPOSIN);-   5) cytotoxic antibiotics, including but not limited to actinomycin D    (dactinomycin, COSMEGEN), bleomycin (BLENOXANE) doxorubicin    (ADRIAMYCIN), daunorubicin (CERUBIDINE), epirubicin (ELLENCE),    fludarabine (FLUDARA), idarubicin, mitomycin (MITOSOL), mitoxantrone    (NOVANTRONE), plicamycin;-   6) aromatase inhibitors, including but not limited to    aminoglutethimide, anastrozole (ARIMIDEX), letrozole (FEMARA),    vorozole (RIVIZOR), exemestane (AROMASIN);-   7) angiogenesis inhibitors, including but not limited to genistein,    sunitinib (SUTENT) and bevacizumab (AVAS TIN);-   8) anti-steroids and anti-androgens such as aminoglutethimide    (CYTADREN), bicalutamide (CASODEX), cyproterone, flutamide    (EULEXIN), nilutamide (NILANDRON);-   9) tyrosine kinase inhibitors, including but not limited to imatinib    (GLEEVEC), erlotinib (TARCEVA), lapatininb (TYKERB), sorafenib    (NEXAVAR), and axitinib (INLYTA);-   10) mTOR inhibitors such as everolimus, temsirolimus (TORISEL), and    sirolimus;-   11) monoclonal antibodies such as trastuzumab (HERCEPTIN) and    rituximab (RITUXAN);-   12) other agents, such as amsacrine; Bacillus Calmette-Guérin    (B-C-G) vaccine; buserelin (ETILAMIDE); chloroquine (ARALEN);    clodronate, pamidronate, and other bisphosphonates; colchicine;    demethoxyviridin; dichloroacetate; estramustine; filgrastim    (NEUPOGEN); fludrocortisone (FLORINEF); goserelin (ZOLADEX);    interferon; leucovorin; leuprolide (LUPRON); levamisole; lonidamine;    mesna; metformin; mitotane (o,p′-DDD, LYSODREN); nocodazole;    octreotide (SANDOSTATIN); perifosine; porfimer (particularly in    combination with photo- and radiotherapy); suramin; tamoxifen;    titanocene dichloride; tretinoin; anabolic steroids such as    fluoxymesterone (HALOTESTIN); estrogens such as estradiol,    diethylstilbestrol (DES), and dienestrol; progestins such as    medroxyprogesterone acetate (MPA) and megestrol; and testosterone.

Where a subject is suffering from or at risk of suffering from aninflammatory condition, a GLS1 inhibitor compound described herein isoptionally used together with one or more agents or methods for treatingan inflammatory condition in any combination. Therapeuticagents/treatments for treating an autoimmune and/or inflammatorycondition include, but are not limited to any of the following examples:

-   1) corticosteroids, including but not limited to cortisone,    dexamethasone, and methylprednisolone;-   2) nonsteroidal anti-inflammatory drugs (NSAIDs), including but not    limited to ibuprofen, naproxen, acetaminophen, aspirin, fenoprofen    (NALFON), flurbiprofen (ANSAID), ketoprofen, oxaprozin (DAYPRO),    diclofenac sodium (VOLTAREN), diclofenac potassium (CATAFLAM),    etodolac (LODINE), indomethacin (INDOCIN), ketorolac (TORADOL),    sulindac (CLINORIL), tolmetin (TOLECTIN), meclofenamate (MECLOMEN),    mefenamic acid (PONSTEL), nabumetone (RELAFEN) and piroxicam    (FELDENE);-   3) immunosuppressants, including but not limited to methotrexate    (RHEUMATREX), leflunomide (ARAVA), azathioprine (IMURAN),    cyclosporine (NEORAL, SANDIMMUNE), tacrolimus and cyclophosphamide    (CYTOXAN);-   4) CD20 blockers, including but not limited to rituximab (RITUXAN);-   5) Tumor Necrosis Factor (TNF) blockers, including but not limited    to etanercept (ENBREL), infliximab (REMICADE) and adalimumab    (HUMIRA);-   6) interleukin-1 receptor antagonists, including but not limited to    anakinra (KINERET);-   7) interleukin-6 inhibitors, including but not limited to    tocilizumab (ACTEMRA);-   8) interleukin-17 inhibitors, including but not limited to AIN457;-   9) Janus kinase inhibitors, including but not limited to    tasocitinib; and-   10) syk inhibitors, including but not limited to fostamatinib.    Compound Synthesis

Compounds of the present invention can be prepared using methodsillustrated in general synthetic schemes and experimental proceduresdetailed below. General synthetic schemes and experimental proceduresare presented for purposes of illustration and are not intended to belimiting. Starting materials used to prepare compounds of the presentinvention are commercially available or can be prepared using routinemethods known in the art.

LIST OF ABBREVIATIONS

Ac₂O=acetic anhydride; AcCl=acetyl chloride; AcOH=acetic acid;AIBN=azobisisobutyronitrile; aq.=aqueous;BAST=bis(2-methoxyethyl)aminosulfur trifluoride; Bu₃SnH=tributyltinhydride; CD₃OD=deuterated methanol; CDCl₃=deuterated chloroform;CDI=1,1′-Carbonyldiimidazole; DAST=(diethylamino)sulfur trifluoride;DBU=1,8-diazabicyclo[5.4.0]undec-7-ene; DCM=dichloromethane;DEAD=diethyl azodicarboxylate; DIBAL-H=di-iso-butyl aluminium hydride;DIEA=DIPEA=N,N-diisopropylethylamine; DMAP=4-dimethylaminopyridine;DMF=N,N-dimethylformamide; DMSO-d₆=deuterated dimethyl sulfoxide;DMSO=dimethyl sulfoxide; DPPA=diphenylphosphoryl azide;EDC.HCl=EDCI.HCl=1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride; Et₂O=diethyl ether; EtOAc=ethyl acetate; EtOH=ethanol;h=hour; HATU=2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium; HMDS=hexamethyldisilazane;HOBT=1-hydroxybenzotriazole; i-PrOH=isopropanol; LAH=lithiumaluminiumhydride; LDA=lithium diisopropyl amide; LiHMDS=Lithiumbis(trimethylsilyl)amide; MeCN=acetonitrile; MeOH=methanol; MP-carbonateresin=macroporous triethylammonium methylpolystyrene carbonate resin;MsCl=mesyl chloride; MTBE=methyl tertiary butyl ether;n-BuLi=n-butyllithium; NaHMDS=Sodium bis(trimethylsilyl)amide;NaOMe=sodium methoxide; NaOtBu=sodium t-butoxide;NBS=N-bromosuccinimide; NCS=N-chlorosuccinimide;NMP=N-Methyl-2-pyrrolidone;Pd(Ph3)₄=tetrakis(triphenylphosphine)palladium(0);Pd₂(dba)₃=tris(dibenzylideneacetone)dipalladium(0);PdCl₂(PPh₃)₂=bis(triphenylphosphine)palladium(II) dichloride;PG=protecting group; prep-HPLC=preparative high-performance liquidchromatography; PMBCl=para-methoxybenzyl chloride;PyBop=(benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate; Pyr=pyridine; RT=room temperature;RuPhos=2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl;sat.=saturated; ss=saturated solution; t-BuOH=tert-butanol;T3P=Propylphosphonic Anhydride; TEA=Et₃N=triethylamine;TFA=trifluoroacetic acid; TFAA=trifluoroacetic anhydride;THF=tetrahydrofuran; Tot=toluene; TsCl=tosyl chloride;Xantphos=4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene;X-Phos=2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl.

General Methods for Preparing Compounds

The following schemes can be used to practice the present invention.Additional structural groups, including but not limited to those definedelsewhere in the specification and not shown in the compounds describedin the schemes can be incorporated to give various compounds disclosedherein, or intermediate compounds which can, after further manipulationsusing techniques known to those skilled in the art, be converted tocompounds of the present invention. For example in certain embodimentsthe A-ring in the structures described in the schemes—wherein A is aheteroaromatic ring—can be substituted with various groups as definedherein.

Non-limiting examples include the following compounds andpharmaceutically acceptable salts thereof.

Example 1:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide

Step 1: 3,6-diiodopyridazine

A mixture of 3,6-dichloropyridazine (60.00 g, 402.7 mmol) and 55%aqueous hydrogen iodide solution (51.51 g, 402.7 mmol, 30.30 mL) wasstirred at 90° C. for 12 h. Solid was isolated by filtration and thensuspended in a sat. aq. NaHCO₃ solution (300 mL). Solid was isolated byfiltration, washing with petroleum ether (2×200 mL), to give the titlecompound as a yellow solid, which was used without further purification(120.0 g, 90%). MS (ES⁺) C₄H₂I₂N₂ requires: 332, found: 333 [M+H]⁺.

Step 2: di-tert-butyl 2-(6-iodopyridazin-3-yl)malonate

To a suspension of NaH (27.12 g, 678.0 mmol, 60% in mineral oil) in THF(750 mL) were added di-tert-butyl propanedioate (97.75 g, 452.0 mmol,100.8 mL) and the mixture was stirred at 28° C. for 15 min.3,6-diiodopyridazine (75.00 g, 225.99 mmol) was added, and the reactionmixture was stirred at reflux for 8 h. The reaction mixture was quenchedwith a sat. aq. NH₄Cl solution (500 mL) and extracted with 1:1EtOAc/petroleum ether (3×500 mL). The combined organic layers were driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by SiO₂ gel chromoatography (10:1 petroleumether/EtOAc) to give the title compound as a white solid (83.00 g, 87%).MS (ES⁺) C₁₅H₂₁IN₂O₄ requires: 420, found: 421 [M+H]⁺.

Step 3: (R)-1-azido-3-(benzyloxy)propan-2-ol

To a solution of (R)-2-((benzyloxy)methyl)oxirane (2.423 ml, 15.89 mmol)and NH₄Cl (1.70 g, 31.8 mmol) in MeOH (39.5 ml) and water (5.92 ml) wasadded sodium azide (5.17 g, 79.0 mmol) and the resulting mixture wasstirred at RT overnight. The mixture was concentrated under reducedpressure and the residue was partitioned between EtOAc (50 mL) and water(60 mL). The two layers were separated, and the aqueous layer wasextracted with EtOAc (3×50 mL). The organic layers were combined, driedover MgSO₄, filtered, and concentrated under reduced pressure to givethe title compound as a colorless oil (3.01 g, 91%). MS (ES⁺) C₁₀H₁₃N₃O₂requires: 207, found: 208 [M+H]⁺.

Step 4: (R)-tert-butyl1-(3-(benzyloxy)-2-hydroxypropyl)-1H-1,2,3-triazole-4-carboxylate

To a solution of (R)-1-azido-3-(benzyloxy)propan-2-ol (3.01 g, 14.5mmol),tert-butyl propiolate (2.393 ml, 17.43 mmol), DIEA (0.253 ml, 1.45mmol), and AcOH (0.083 ml, 1.45 mmol) in DCM (58.1 ml) was added CuI(0.138 g, 0.726 mmol) and the resulting mixture was stirred at RTovernight. SiO₂ gel (10 g) was added to the stirring mixture and theresulting suspension was filtered and washed with DCM (20 mL) and EtOAc(20 mL). The filtrate was concentrated under reduced pressure to givecrude title compound as an orange oil, which was used without furtherpurification (3.95 g, 82%). MS (ES⁺) C₁₇H₂₃N₃O₄ requires: 333. found:334 [M+H]⁺.

Step 5: (S)-tert-butyl1-(3-(benzyloxy)-2-fluoropropyl)-1H-1,2,3-triazole-4-carboxylate

To a 0° C. solution of (R)-tert-butyl1-(3-(benzyloxy)-2-hydroxypropyl)-1H-1,2,3-triazole-4-carboxylate (3.95g, 11.8 mmol) and pyridine (1.909 ml, 23.70 mmol) in DCM (23.70 ml) wasadded DAST (3.13 ml, 23.7 mmol). The resulting mixture was stirred at RTfor 2.5 h, then filtered through a plug of SiO₂ gel, rinsing with DCM(50 mL). The filtrate was concentrated under reduced pressure and theresidue was adsorbed onto Celite and purified by SiO₂ chromatography (0to 50% EtOAc in hexanes) to give the title compound as a tan crystallinesolid (1.781 g, 45% yield). MS (ES⁺) C₁₇H₂₂FN₃O₃ requires: 335, found:336 [M+H]⁺.

Step 6: (S)-tert-butyl1-(2-fluoro-3-hydroxypropyl)-1H-1,2,3-triazole-4-carboxylate

A reaction vessel was charged with (S)-tert-butyl1-(3-(benzyloxy)-2-fluoropropyl)-1H-1,2,3-triazole-4-carboxylate (1.78g, 5.31 mmol) and EtOAc (53.1 ml) under an atmosphere of N₂. Thesolution was purged with N₂ for 10 min. and then with N₂ still flowing,Pd(OH)₂ on carbon (0.746 g, 1.06 mmol) was added. The resultingsuspension was stirred as it was purged with H₂ for 2 min. The reactionmixture was stirred under an atmosphere of H₂ at 1 atm for 12 h, thenpurged with N₂, filtered through Celite and concentrated under reducedpressure to give the title compound as pale yellow crystals (1.32 g,101% yield). MS (ES⁺) C₁₀H₁₆FN₃O₃ requires: 245, found: 246 [M+H]⁺.

Step 7: (S)-tert-butyl1-(2-fluoro-3-(tosyloxy)propyl)-1H-1,2,3-triazole-4-carboxylate

To a solution of (S)-tert-butyl1-(2-fluoro-3-hydroxypropyl)-1H-1,2,3-triazole-4-carboxylate (1.32 g,5.38 mmol) and DMAP (0.986 g, 8.07 mmol) in DCM (26.9 ml) was added4-methylbenzene-1-sulfonyl chloride (1.23 g, 6.46 mmol) while thesolution was maintained at RT by a water bath. The resulting mixture wasstirred at RT for 1.5 h, then diluted with EtOAc (100 mL) and washedwith sat. aq. NH₄Cl (2×40 mL). The organic layer was dried over MgSO₄,filtered and concentrated under reduced pressure to give the crude titlecompound, which was used without further purification (1.803 g, 84%). MS(ES⁺) C₁₇H₂₂FN₃O₅S requires: 399, found: 400 [M+H]⁺.

Step 8: (S)-tert-butyl1-(2-fluoro-3-iodopropyl)-1H-1,2,3-triazole-4-carboxylate

To a solution of (S)-tert-butyl1-(2-fluoro-3-(tosyloxy)propyl)-1H-1,2,3-triazole-4-carboxylate (2.12 g,5.31 mmol) in acetone (26.5 ml) was added sodium iodide (0.796 g, 5.31mmol) and the resulting mixture was stirred at 80° C. for 3 h.Additional sodium iodide (1.6 g) was added and the mixture as stirred at90° C. for 2 h. The mixture was allowed to cool to RT, then diluted with1:1 EtOAc/hexanes (150 mL) and sequentially washed with H₂O (2×50 mL)and a sat. aq. NaCl solution (50 mL). The organic layer was dried overMgSO₄, filtered, and concentrated under reduced pressure. The residuewas adsorbed onto Celite and purified by SiO₂ gel chromatography (0 to50% EtOAc/hexanes) to give the title compound as a white crystallinesolid (1.71 g, 91%). MS (ES⁺) C₁₀H₁₅FIN₃O₂ requires: 355, found: 356[M+H]⁺.

Step 9: (R)-di-tert-butyl2-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-iodopyridazin-3-yl)malonate

A mixture of potassium carbonate (0.412 g, 2.98 mmol), di-tert-butyl2-(6-iodopyridazin-3-yl)malonate (1.25 g, 2.98 mmol), and (S)-tert-butyl1-(2-fluoro-3-iodopropyl)-1H-1,2,3-triazole-4-carboxylate (1.00 g, 2.82mmol) in a vial was degassed and then DMF (9.39 ml) was added. Themixture was degassed and backfilled with nitrogen for three cycles andthen stirred for 80 hours at 25° C. The mixture was diluted with ethylacetate and hexanes (1:1, 200 mL) and washed with water twice (100mL+100 mL). The combined aqueous layer was extracted with ethyl acetatehexanes (1:1, 100 mL). The combined organic layer was concentrated, wasadded brine and the organic layer was concentrated under reducedpressure. The residue was purified by SiO₂ gel chromatography (5 to 60%EtOAc in hexanes) to give the title compound (1.36 g, 74.6% yield) as ayellow liquid. MS (ES+) C₂₅H₃₅FIN₅O₆ requires: 647, found: 648 [M+H]⁺.

Step 10: 2,6-dichloro-4-(3,3-difluorocyclobutoxy)pyridine

To a suspension of NaH (8.88 g, 60% in mineral oil, 222 mmol) in THF(800 ml) at 0° C. was added 3,3-difluorocyclobutanol (20 g, 185 mmol)dropwise over a period of 10 min. Right after the completion of addition(bubbling should have quickly stopped), 2,6-dichloro-4-nitropyridine(35.7 g, 185 mmol) was added portionwise and the resulting mixture wasstirred at 0° C. for 1 h. Sat. aq. NH₄Cl (200 mL) and water (800 ml)were added, and the layers were separated. The aqueous phase wasextracted with EtOAc (3×500 mL), and the combined organic layers werewashed with sat. aq. NaCl, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by SiO₂ gelchromatography (0 to 8% EtOAc in hexanes) to give the title compound asa white crystalline solid (45.0 g, 96%). MS (ES⁺) C₉H₇Cl₂F₂NO requires:253, found: 254 [M+H]⁺.

Step 11: 2-chloro-4-(3,3-difluorocyclobutoxy)-6-methylpyridine

To a solution of 2,6-dichloro-4-(3,3-difluorocyclobutoxy)pyridine (45 g,177 mmol), THF (800 ml), NMP (200 ml) and ferric acetylacetonate (1.877g, 5.31 mmol) at 0° C. was added dropwise methylmagnesium bromide (3 Min ether, 77 ml, 230 mmol) and the resulting mixture was stirred at 0°C. for 0.5 h. The reaction was quenched with sat. aq. NH₄Cl (100 mL) at0° C., water (900 ml) was added, and the layers were separated. Theaqueous phase was extracted with EtOAc (3×500 mL), and the combinedorganic layers were washed with sat. aq. NaCl, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by SiO₂ gel chromatography (0 to 20% EtOAc in hexanes) to givethe title compound as a colorless liquid (36.5 g, 88%). MS (ES⁺)C₁₀H₁₀ClF₂NO requires: 233, found: 234 [M+H]⁺.

Step 12: ethyl2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetate

A degassed solution of2-chloro-4-(3,3-difluorocyclobutoxy)-6-methylpyridine (33.0 g, 141mmol), (2-ethoxy-2-oxoethyl)zinc(II) bromide (0.5 M in THF, 706 ml, 353mmol), Pd2(dba)3 (6.47 g, 7.06 mmol) and XPhos (3.37 g, 7.06 mmol) wasstirred at 50° C. for 1 h. The reaction mixture was allowed to cool toRT and sat. aq. NH₄Cl (100 mL) and water (900 mL) were added.Precipitate was removed by filtration, and the filtrate layers wereseparated. The aqueous phase was extracted with EtOAc (3×500 mL), andthe combined organic layers were washed with sat. aq. NaCl, dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by SiO₂ gel chromatography (0 to 60% EtOAc in hexanes) to givethe title compound as a yellow liquid (27.8 g, 69%). MS (ES⁺)C₁₄H₁₇F₂NO₃ requires: 285, found: 286 [M+H]⁺.

Step 13: 2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamide

A solution of ethyl2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetate (27.8 g,97.0 mmol) and NH₃/MeOH (7 M, 557 ml, 3898 mmol) in a pressure bottlewas stirred at 85° C. for 20 h. The reaction mixture was allowed to coolto RT, then concentrated under reduced pressure. The resulting solid wastriturated with ether and isolated by filtration to give the titlecompound as an off-white solid (22.4 g, 90%). MS (ES⁺) C₁₂H₁₄F₂N₂O₂requires: 256, found: 257 [M+H]⁺.

Step 14: di-tert-butyl(R)-2-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)malonate

A degassed solution of (R)-di-tert-butyl2-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-iodopyridazin-3-yl)malonate(42.4 g, 65.6 mmol),2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamide (14.0 g,54.6 mmol), cesium carbonate (35.6 g, 109 mmol), Xantphos (6.32 g, 10.9mmol) and allylpalladium chloride dimer (1.00 g, 2.73 mmol) in dioxane(300 ml) was stirred at 70° C. for 16 h. The reaction mixture wasallowed to cool to RT, then filtered and the filtrate was concentratedunder reduced pressure. The residue was purified by SiO₂ gelchromatography (0 to 3% MeOH in DCM) to give the title compound (36.5 g,86%) as a foamy yellow solid. MS (ES⁺) C₃₇H₄₈F₃N₇O₈ requires: 775,found: 776 [M+H]⁺.

Step 15: (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-1H-1,2,3-triazole-4-carboxylic acid

A solution of (R)-di-tert-butyl2-(3-(4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2-fluoropropyl)-2-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)malonate(36.0 g, 46.4 mmol) in an HCl in dioxane solution (4.0 M, 696.0 ml, 2784mmol) was stirred at 70° C. for 16 h. White precipitate formed. Thereaction mixture was allowed to cool to RT. Precipitate was isolated byfiltration, washed with EtOAc, and dried in vacuo to give the titlecompound as an off-white solid, which was used without furtherpurification in the next step. MS (ES⁺) C₂₃H₂₄F₃N₇O₄ requires: 519,found: 520 [M+H]⁺.

Step 16:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide

To a solution of crude(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-1H-1,2,3-triazole-4-carboxylicacid hydrochloride prepared in the previous step (assumed 46.4 mmol) inDMF (200 ml) at 0° C. was added HATU (17.64 g, 46.4 mmol), DIEA (40.5ml, 232 mmol) and methanamine in THF (2.0 M, 27.8 ml, 55.7 mmol) and theresulting mixture was stirred at 20° C. for 1 h. The volatiles wereremoved under reduced pressure. Water (1000 mL) and DCM (500 ml) wereadded, and the layers were separated. The aqueous phase was extractedwith DCM (3×300 mL), the combined organic layers were washed with sat.aq. NaCl, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by SiO₂ gel chromatography (0 to 8%MeOH in DCM) to give the title compound (16.8 g, 68.0% yield) as a whitesolid. MS (ES+) C₂₄H₂₇F₃N₈O₃ requires: 532, found: 533 [M+H]⁺. ¹H NMR(DMSO-d₆) δ 11.30 (s, 1H), 8.51 (s, 1H), 8.47 (q, J=4.4, 1H), 8.22 (d,J=9.1 Hz, 1H), 7.60 (d, J=9.3 Hz, 1H), 6.79 (d, J=2.5 Hz, 1H), 6.72 (d,J=2.5 Hz, 1H), 5.09-4.96 (m, 1H), 4.90-4.70 (m, 3H), 3.87 (s, 2H),3.28-3.18 (m, 2H), 3.08-2.98 (m, 2H), 2.76 (d, J=4.9 Hz, 3H), 2.75-2.63(m, 2H), 2.39 (s, 3H), 2.20-1.95 (m, 2H).

Step 17:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidedihydrochloride

To a solution of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(12.71 g, 23.87 mmol) in MeOH (20 ml) and DCM (60 ml) at 0° C. was addedHCl in dioxane (4.0 M, 11.93 ml, 47.70 mmol) and the resulting mixturewas stirred for 5 min. then concentrated under reduced pressure. Theresidue was redissolved in MeCN and water, lyophilized, and theresulting solid was triturated with EtOAc and dried in vacuo to give thetitle compound (14.03 g, 97%) as a white solid. MS (ES⁺) C₂₄H₂₇F₃N₈O₃requires: 532, found: 533 [M+H]⁺. ¹H NMR (DMSO-d₆) δ 11.66 (s, 1H), 8.53(s, 1H), 8.47 (q, J=5.3, 1H), 8.23 (d, J=9.1 Hz, 1H), 7.73 (d, J=9.5 Hz,1H), 7.44 (d, J=2.0 Hz, 1H), 7.39 (d, J=2.0 Hz, 1H), 4.96-5.11 (m, 2H),4.67-4.86 (m, 2H), 4.36 (s, 2H), 3.34 (m, 2H), 3.07 (m, 2H), 2.87 (m,2H), 2.76 (d, J=4.9 Hz, 3H), 2.68 (s, 3H), 2.24-1.95 (m, 2H). The titlecompound (2 mg/mL, 10 μL per injection) was analyzed on a ShimadzuProminence HPLC system with a Lux Cellulose 4 column (4.6×150millimeter, 5 micrometer, 1 mL/min) using a mobile phase ofwater:acetonitrile (50:50), and showed an ee of >98%. Retention time:11.3 mins.

Example 1 disclosed above may also be made by Scheme 2.

Example 2:(S)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidedihydrochloride

Made in the same fashion as Example 1. MS (ES⁺) C₂₄H₂₇F₃N₈O₃ requires:532, found: 533 [M+H]⁺. The title compound (2 mg/mL, 10 μL perinjection) was analyzed on a Shimadzu Prominence HPLC system with a LuxCellulose 4 column (4.6×150 millimeter, 5 micrometer, 1 mL/min) using amobile phase of water:acetonitrile (50:50), and showed an ee of >98%.Retention time: 9.3 mins.

Example 3:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidehydrochloride

To a solution of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(20 mg, 0.038 mmol) in dioxane (1.8 mL) at RT was added aqueous HCl(37.6 μl, 0.038 mmol, 1.00 M) drop wise. A whitish precipitate formedimmediately. The resulting mixture was stirred at RT for 15 min. Themixture was diluted with Et₂O (1.8 mL), cooled to 0° C., and thesupernatant removed. The remaining solid was diluted with water (2 mL)to form a clear solution and lyophilized to obtain the title compound asa white solid (19 mg, 89%). MS (ES+) C₂₄H₂₇F₃N₈O₃.HCl requires: 532,found: 533 [M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆) δ 15.20 (br s, 1H), 11.55(s, 1H), 8.52 (s, 1H), 8.50-8.44 (m, 1H), 8.17 (d, J=9.1 Hz, 1H), 7.64(d, J=9.2 Hz, 1H), 7.38 (d, J=21.5 Hz, 2H), 5.11-4.95 (m, 2H), 4.86-4.69(m, 2H), 4.29 (s, 2H), 3.37-3.30 (m, 2H), 3.12-2.99 (m, 2H), 2.91-2.80(m, 2H), 2.76 (d, J=4.5 Hz, 3H), 2.66 (s, 3H), 2.21-2.07 (m, 1H),2.07-1.96 (m, 1H).

Example 4:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidesulfate

Prepared using the procedure in Example 3 to give the title compound asa white solid (89%). MS (ES+) C₂₄H₂₇F₃N₈O₃.H₂SO₄ requires: 532, found:533 [M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆) δ 14.70 (br s, 1H), 11.55 (s, 1H),8.52 (s, 1H), 8.50-8.43 (m, 1H), 8.17 (d, J=9.1 Hz, 1H), 7.65 (d, J=9.1Hz, 1H), 7.39 (d, J=14.5 Hz, 2H), 5.12-4.95 (m, 2H), 4.87-4.70 (m, 2H),4.24 (s, 2H), 3.39-3.28 (m, 2H), 3.11-2.99 (m, 2H), 2.92-2.81 (m, 2H),2.76 (d, J=4.6 Hz, 3H), 2.65 (s, 3H), 2.22-2.08 (m, 1H), 2.08-1.92 (m,1H).

Example 5:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidemethanesulfonate

Prepared using the procedure in Example 3 using MeSO₃H (1.0 M in CH₂Cl₂)to give the title compound as a white solid (93%). MS (ES+)C₂₄H₂₇F₃N₈O₃.CH₃SO₃H requires: 532, found: 533 [M+H]⁺. ¹H NMR (600 MHz,DMSO-d₆) δ 14.71 (br s, 1H), 11.53 (s, 1H), 8.52 (s, 1H), 8.50-8.44 (m,1H), 8.17 (d, J=9.1 Hz, 1H), 7.64 (d, J=9.1 Hz, 1H), 7.34 (d, J=17.3 Hz,2H), 5.12-4.96 (m, 2H), 4.86-4.68 (m, 2H), 4.21 (s, 2H), 3.34-3.28 (m,2H), 3.10-3.00 (m, 2H), 2.91-2.80 (m, 2H), 2.76 (d, J=4.6 Hz, 3H), 2.63(s, 3H), 2.30 (s, 3H), 2.21-2.07 (m, 1H), 2.07-1.94 (m, 1H).

Example 6:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidehydrobromide

Prepared using the procedure in Example 3 to give the title compound asa white solid (87%). MS (ES+) C₂₄H₂₇F₃N₈O₃.HBr requires: 532, found: 533[M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆) δ 14.76 (br s, 1H), 11.56 (s, 1H),8.52 (s, 1H), 8.50-8.43 (m, 1H), 8.17 (d, J=9.2 Hz, 1H), 7.65 (d, J=9.2Hz, 1H), 7.41 (d, J=15.7 Hz, 2H), 5.11-4.97 (m, 2H), 4.86-4.70 (m, 2H),4.26 (s, 2H), 3.39-3.29 (m, 2H), 3.11-2.99 (m, 2H), 2.92-2.80 (m, 2H),2.76 (dd, J=4.8, 1.2 Hz, 3H), 2.66 (s, 3H), 2.22-2.08 (m, 1H), 2.08-1.93(m, 1H).

Example 7:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide4-methylbenzenesulfonate

Prepared using the procedure in Example A using aqueous TsOH (1.0 M) togive the title compound as a white solid (86%). MS (ES+)C₂₄H₂₇F₃N₈O₃.TsOH requires: 532, found: 533 [M+H]⁺. ¹H NMR (600 MHz,DMSO-d₆) δ 14.69 (br s, 1H), 11.54 (s, 1H), 8.52 (s, 1H), 8.49-8.44 (m,1H), 8.17 (d, J=9.1 Hz, 1H), 7.64 (d, J=9.1 Hz, 1H), 7.47 (d, J=8.0 Hz,2H), 7.37 (d, J=15.9 Hz, 2H), 7.10 (dd, J=8.1, 1.0 Hz, 2H), 5.12-4.94(m, 2H), 4.88-4.68 (m, 2H), 4.23 (s, 2H), 3.37-3.27 (m, 2H), 3.10-2.99(m, 2H), 2.92-2.80 (m, 2H), 2.76 (d, J=4.7 Hz, 3H), 2.64 (s, 3H), 2.28(s, 3H), 2.22-2.07 (m, 1H), 2.07-1.94 (m, 1H).

To obtain a crystalline solid, the title compound (5.0 mg) was stirredas a slurry in EtOH (200 uL) in a sealed vial for 48 h. The suspensioninitially dissolved and then slowly reformed small, needle-like crystalsas observed under an optical microscope. The mixture was concentratedand the crystalline solid analyzed by HNMR, which was identical to theparent compound.

Example 8:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamidedihydrochloride

To a solution of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(5.00 g, 9.39 mmol) in MeOH (5 mL) and DCM (15 mL) at 0° C. was addedHCl (4.69 ml, 18.7 mmol, 4 M in dioxane) and the resulting mixture wasstirred for 5 min. The volatiles were removed under reduced pressure.The residue was re-dissolved in MeCN and water and lyophilized to givethe title compound (5.69 g, 100%) as a white solid. MS (ES+)C₂₄H₂₇F₃N₈O₃.2HCl requires: 532, found: 533 [M+H]⁺. ¹H NMR (600 MHz,DMSO-d₆) δ 14.85 (br s, 1H), 11.55 (s, 1H), 8.52 (s, 1H), 8.47 (m, 1H),8.17 (d, J=9.1 Hz, 1H), 7.64 (d, J=9.1 Hz, 1H), 7.39 (d, J=12.8 Hz, 2H),5.11-4.97 (m, 2H), 4.85-4.71 (m, 2H), 4.25 (s, 2H), 3.38-3.29 (m, 2H),3.10-3.00 (m, 2H), 2.91-2.81 (m, 2H), 2.76 (d, J=4.7 Hz, 3H), 2.65 (s,3H), 2.20-1.96 (m, 2H).

Example 9: Polymorphs of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide

Polymorph A: The title compound (5.00 mg, 9.39 μmol) was suspended in 50uL of 20% water in acetone (v/v), and the mixture was heated at 95° C.for 10 min. The suspension became a clear solution and the sample wasleft standing at RT to crystallize. The solids formed were observedunder an optical microscope and found to be very fine, hair-likeneedles.

Polymorph B: The title compound (5.00 mg, 9.39 μmol) was suspended in 50uL of 10% water in DMSO (v/v), and the mixture was heated at 95° C. for10 min. The suspension became a clear solution and the sample was leftstanding at RT to crystallize. The solids formed were observed under anoptical microscope and found to be rod-like crystals.

Polymorph C: The title compound (5.00 mg, 9.39 μmol) was suspended in100 uL of 40% ethanol in anisole (v/v), and the mixture was heated at95° C. for 10 min. The suspension became a clear solution and the samplewas left standing at RT to crystallize. The solids formed were observedunder an optical microscope and found to be fine, plate-like.

Example 10:(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide1:1 DMSO Solvate

A suspension of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(100 mg, 0.188 mmol) in 1 mL of 15% water in DMSO (v/v) was heated at95° C. for 1 min. The resulting clear solution was cooled to 80° C. andstirred for 10 min. The stirring was stopped and the mixture was allowedto cool by 10° C. every 20 min until the temperature reached 40° C. Themixture was then allowed to cool to RT overnight. The mixture wasdiluted with 1 mL of acetone, vortexed very briefly to breakdown thesuspension, and filtered. The collected solids were rinsed with acetone(2×1 mL). The solid was dried under high vacuum overnight to obtain thetitle compound as a clear, crystalline solid (86 mg, 79%). This materialappears as rod-like crystals under an optical microscope. MS (ES⁺)C₂₄H₂₇F₃N₈O₃.DMSO requires: 532, found: 533 [M+H]⁺. ¹H NMR (600 MHz,CD₃OD) δ 8.36 (d, J=9.1 Hz, 1H), 8.34 (s, 1H), 7.61 (d, J=9.2 Hz, 1H),6.79 (d, J=1.9 Hz, 1H), 6.72 (d, J=1.9 Hz, 1H), 4.90-5.15 (m, 1H),4.68-4.83 (m, 3H), 3.91 (s, 2H), 3.01-3.23 (m, 4H), 2.92 (s, 3H),2.67-2.78 (m, 2H), 2.65 (s, 6H), 2.49 (s, 3H), 2.24-1.99 (m, 2H).

Physical Characterization of Example 1 Free Base and Salts

The Example 1 free base and salts were characterized by XRPD, DSC, TGA,PLM and DVS. The detailed procedures are listed below:

X-ray Powder Diffraction (XRPD) About 10 mg of test compound was weighedand distributed evenly onto a single crystal silicon plate. The samplewas rotated at 10°/min. The diffraction pattern was measured with a witha Bruker D8 ADVANCE using a CuKα source (λ=1.54179 Å) operating at 40 KVof tube voltage and 40 mA of tube current. The scattering angle wasscanned from 2θ=3° to 40° at a step rate of 10°/min.

Thermogravimetric Analysis (TGA) TGA was performed on a TA InstrumentsQ5000IR instrument. About 5 mg of test compound was weighed andtransferred to an aluminum oxide crucible. The sample was heated from RTto 400° C. at a rate of 10° C./min under 50 mL/min N₂ purge.

Differential Scanning calorimetry (DSC) DSC was performed on a TAInstruments Q2000 device. About 1 mg of test compound was weighed andtransferred to a crimped aluminum pans with pinhole. The sample washeated from 30° C. to 400° C. at a rate of 10° C./min.

Polarized Light Microscope (PLM) An amount of the test compound as apowder was dispersed in silicone oil, and the morphology was examinedwith a Nikon LV100POL equipped with 5 megapixel CCD, 10× ocular lens,and objective lens chosen from 20× and 50×. The appropriate objectivelens was chosen for the sample under examination.

Infrared Spectrometry (IR) was performed on a Thermo Nicolet 380 FT-IRspectrometer, using a DTGS detector with KBr windows and a Ge on KBrbeamsplitter. 32 scans, having 4 cm⁻¹ resolution and with wavelengthrange of 4000 to 400 cm⁻¹ were collected and averaged.

High Performance Liquid Chromatography (HPLC) was run on an Agilent 1200series HPLC instrument, equipped with a Amide_80, TSK_Gel (4.6 mm*150mm, 3 μm) column. Mobile phase consisted of an 80:20 mix of solvent“A”:solvent “B”, wherein “A” was 85% CH₃CN:15% aq NH₄OAc, and “B” was10% CH₃CN:90% aq NH₄OAc. Flow rate was 1.0 mL/min, at 25° C., for atotal run time of 10 min. Detection was accomplished with an ELSD (SEDEX85, 45° C., 3.5 bar, Nitrogen) detector.

¹H Nuclear Magnetic Resonance (¹H NMR) was recorded on a Bruker AVANCEIII with manual phasing, pulse width 300 msec, acquisition time of 3.98sec with 1 sec relaxation delay, time domain 24 K, 8 transientscollected, and exponential multiplication of 0.5 for line broadening.

Dynamic Vapor Sorption (DVS) was examined with an SMS Advantageinstrument. About 10 mg of sample was transferred into the instrument,and the weight change with respect to the atmospheric humidity at 25° C.was recorded accordingly, with the following parameters: Equilibrium:dm/dt: 0.01%/min. (for min: 10 min and max: 180 min); drying: 0% RH for120 min; RH (%) measurement step: 10%; RH (%) measurement step scope:0-90-0%.

As determined by XRPD, Example 1 free base was not fully crystalline andpossibly contained partial amorphous content. PLM displayedbirefringence phenomena and irregular block like shape. Also, DSC showedan endothermic peak with onset at 196° C. which was attributed tomelting, An approximate 0.9% weight loss from 30° C. to 200° C. shown inTGA indicated that Example 1 free base is likely in anhydrous form.

Example 11: Polymorph D of(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide

A preliminary slurry test was conducted to purify the crystal form ofExample 1 free base. About 30 mg of the title compound was weighed andtransferred into a 2 mL glass vial. To the vial was added 1 mL of aselected solvent (acetone, acetonitrile, ethyl acetate, ethanol,methanol, 1:1 methanol:water, and tetrahydrofuran) to achieve ahomogeneous suspension. The obtained suspension was agitated at 40° C.for 1 day on a thermomixer, then centrifuged at a speed of 10000 rpm.The supernatant was discarded to afford platelike crystals (FIG. 3 ).

Based on the XRPD results (FIG. 1 ), the amorphous content was reducedvia this purification procedure in most solvents, particularly inacetone (FIG. 2 ). No obvious thermal dynamic change was found afterrecrystallization in acetone based on TGA-DSC profiles (FIG. 4), DSCshowed a endothermic peak with onset at 197° C. which is attributed tomelting. Loss of approximately 0.6% of from 30° C. to 200° C. shown inTGA indicated that Example 1 Polymorph D is likely in the anhydrousform. FIG. 5 displays the IR spectrum of Polymorph D. FIG. 10 displaysthe ¹H NMR spectrum of Polymorph D.

¹H NMR (400 MHz, methanol-d₄) δ ppm 2.03-2.26 (m, 2H), 2.51 (s, 3H),2.67-2.81 (m, 2H), 2.95 (s, 3H), 3.08-3.30 (m, 4H), 3.94 (s, 2H),4.71-5.05 (m, 4H), 6.75 (d, J=2.01 Hz, 1H), 6.81 (d, J=2.01 Hz, 1H),7.62 (d, J=9.29 Hz, 1H), 8.37 (s, 1H), 8.39 (d, J=8.46 Hz, 1H).

Provided herein is solid Example 1 (e.g. Polymorph D) characterized byhaving NMR peaks as disclosed above. In certain embodiments, solidExample 1 (e.g. Polymorph D) is characterized by having at least one, atleast three, or at least five of the peaks as disclosed above. Incertain embodiments, solid Example 1 (e.g. Polymorph D) is characterizedby having between five and ten of the peaks as disclosed above. Suchpeaks may be referred to by their shift in parts per million.

Provided herein is solid Example 1 (e.g. Polymorph D) characterized byhaving one or more ¹H nuclear magnetic resonance (NMR) chemical shiftsat about 2.0-about 2.3, about 2.5, about 2.7-about 2.8, about 3.0, about3.1-about 3.3, about 3.9, about 4.7-about 5.1, about 6.8, about 7.6, orabout 8.4 parts per million. In certain embodiments, the solid Example 1(e.g. Polymorph D) is characterized by having two, three, four, five, ormore of the shifts. In certain embodiments, the solid Example 1 (e.g.Polymorph D) is characterized by having three or more of the shifts. Incertain embodiments, the solid Example 1 (e.g. Polymorph D) ischaracterized by having five or more of the shifts.

The DVS result (FIG. 7 and Table 1) showed that Example 1 Polymorph Dwas non-hygroscopic with 0.1% water sorption from 0-80% RH, and no formtransformation (FIG. 8 ) was found after DVS.

TABLE 1 DVS of purified free base Example 1 (Polymorph D). Target Changeof Mass % P/P₀ Sorption Desorption Hysteresis 0 0.000 0.052 10 0.02160.0276 0.0060 20 0.0336 0.0440 0.0104 30 0.0474 0.0587 0.0112 40 0.05780.0759 0.0181 50 0.0690 0.0906 0.0216 60 0.0828 0.1087 0.0259 70 0.10350.1294 0.0259 80 0.1337 0.1527 0.0190 90 0.1768 0.1768

TABLE 2 XRPD of Polymorph D. 2-Theta d(Å) BG Height I % Area I % FWHM3.967 22.2531 364 5375 82.4 39115 100.0 0.122 7.932 11.1367 179 130420.0 7483 19.1 0.096 11.584 7.6329 148 4108 63.0 22671 58.0 0.093 11.9127.4234 154 6521 100.0 34719 88.8 0.089 14.956 5.9187 130 2025 31.1 988825.3 0.082 15.899 5.5696 126 5516 84.6 29798 76.2 0.091 17.561 5.0460116 1238 19.0 5968 15.3 0.081 19.893 4.4594 109 5065 77.7 29715 76.00.098 20.222 4.3876 114 1009 15.5 6342 16.2 0.105 22.355 3.9736 110 78012.0 4293 11.0 0.092 23.713 3.7490 111 748 11.5 6584 16.8 0.148 23.9313.7154 107 3814 58.5 23368 59.7 0.103

Provided herein is solid Example 1 (e.g. Polymorph D) characterized byhaving XRPD peaks as disclosed above. In certain embodiments, solidExample 1 (e.g. Polymorph D) is characterized by having at least one, atleast three, or at least five of the XRPD peaks as disclosed above. Incertain embodiments, solid Example 1 (e.g. Polymorph D) is characterizedby having between five and ten of the XRPD peaks as disclosed above.Such peaks may be referred to by their 2 theta shift.

Provided herein is solid Example 1 (e.g. polymorph D), characterized byhaving one or more X-ray powder diffraction peaks chosen from about 4.0,about 8.0, about 11.6, about 11.9, about 14.9, about 15.9, about 17.6,about 19.9, about 20.2, about 22.4, about 23.7, and about 23.9 degrees2-theta. In certain embodiments, the Example 1 (e.g. polymorph D) ischaracterized by having two, three, four, five, or more of the peaks. Incertain embodiments, the Example 1 (e.g. polymorph D) is characterizedby having three or more of the peaks. In certain embodiments, theExample 1 (e.g. polymorph D) is characterized by having five or more ofthe peaks. Also provided is solid Example 1 (e.g. polymorph D),characterized by having a X-ray powder diffraction pattern as shown inFIG. 2 .

Salt Preparation

Salt Preparation Method 1: The acids listed in Table 3 were selected toform salts with Example 1 in MeOH.

Step 1: Approximately 50 mg of Example 1 free base was dissolved in a 8mL glass vial with 5 mL of MeOH to achieve a homogeneous suspension.

Step 2: An amount of solid acid was dissolved in a 8 mL glass vial withMeOH.

Step 3: A volume of acid solution sufficient to obtain a desired molarratio of Example 1:acid (the volume of acid solutions added are listedin Table 3) was slowly titrated into the free base solution on themagnetic stirrer.

Step 4: The obtained liquid phase was stirred at room temperature for 24hours to afford a precipitate, which was isolated by centrifugation.

The solid was analyzed with XRPD to determine if a new crystalline formwas obtained, and then dried under vacuum at 40° C. overnight forfurther characterization. Example 1 and pure solid acid suspended inMeOH were used as XRPD standards.

Results are disclosed in Table 3 and FIG. 9 . Solids obtained fromhydrochloric acid, sulfuric acid, and methanesulfonic acid showeddifferent XRPD diffractograms compared with free base, suggesting theformation of chloride, sulfate, and mesylate salts, respectively. Use ofacids selected from a different group afforded only free base, asdetermined by XRPD (FIG. 10 ).

TABLE 3 Salt preparation using Method 1. Acid solution Volume Molarconcentration, of acid Acid Ratio mg/mL solution Observation XRD patternHCl 1:1 11.8 0.864 Clear, and then 10 times of Amorphous MTBE was addedto produce precipitate H₂SO₄ 1:1 18.4 0.562 Clear, and then 10 times ofAmorphous MTBE was added to produce precipitate H₃PO₄ 1:1 16.85 0.668 Nochange Free base MsOH 1:1 14.8 0.671 Clear, and then 10 times of PoorMTBE was added to produce crystalline precipitate TsOH 1:1 10 1.933Clear, and then 10 times of No solid MTBE was added to produceprecipitate, eventually redissolved Citric 1:1 10 2.171 No change Freebase acid Fumaric 1:1 10 1.199 No change Free base acid Malonic 1:1 101.075 No change Free base acid

Recrystallization of Method 1 Product: Potential salts (chloride,sulfate and mesylate) produced with Method 1 were recrystallized inseveral solvents in order to improve crystallinity.

About 2 mg of salt candidate was weighed into a 1.5 mL glass vial, andthen 0.2 mL of solvent (EtOH, ACN, acetone, EtOAc and THF) was added.The obtained suspension was agitated at 40° C. for 1 day on a thermalmixer, then centrifuged at 10000 rpm. The resulting solid was driedunder vacuum at 40° C. overnight for XRPD analysis.

XRPD results (FIG. 11 , FIG. 12 , FIG. 13 ) demonstrated that thecrystallinity of two of the three salts was improved via thisrecrystallization method in certain solvents: the chloride salt in ACN,the sulfate salt in EtOH.

Salt Preparation Method 2: This method employs a higher concentration ofthe free base, and slow evaporation of sovent, in order to obtain highlycrystalline salt forms. Results with various acids, using EtOH assolvent, are disclosed in Table 4.

Step 1: About 10 mg of Example 1 free base was dissolved in a 1.5 mLglass vial with 0.2 mL of EtOH to achieve a homogeneous suspension.

Step 2: Next, an appropriate amount of solid acid was dissolved in a 8mL glass vial with EtOH.

Step 3: A volume of acid solution sufficient to obtain a desired molarratio of Example 1:acid was slowly titrated into free base solution withstirring on a magnetic stirrer. The volume of acid solutions add arelisted in Table 4.

Step 4: The liquid phase thus obtained was stirred at room temperaturefor 24 hours to afford a precipitate.

The solid that was obtained through centrifugation was analyzed by XRPDto determine if a new crystalline form was obtained, and then driedunder vacuum at 40° C. overnight for further characterization. Example 1and pure solid acid suspended in MeOH were used as standards for Example1 and pure solid acid, respectively.

Based on the results disclosed in Table 4 and FIG. 14 , chloride,sulfate, mesylate, and sulfate salts were potentially formed; howevercrystallinity of these salts was poor.

TABLE 4 Salt preparation using Method 2. Acid solution Volume Molarconcentration, of acid Acid Ratio mg/ml. solution Observation XRDpattern HCl 1:1 59 0.034 Clear, and then white solid Poor precipitatedcrystalline H₂SO₄ 1:1 61.3 0.033 Clear, and then white solid Poorprecipitated crystalline MsOH 1:1 49.3 0.040 Clear, and then white solidPoor precipitated crystalline TsOH 1:1 50 0.078 Clear, and then allowedslow Poor evaporation to afford solid crystalline

Salt Preparation, Method 3: The 3rd round of salt synthesis wasperformed with an optimized procedure to generate highly crystallinesalt forms. The details are listed below:

Step 1: Approximately 100 mg of Example 1 free base was dissolved in a 8mL glass vial at 40° C. with 2 mL of ACN to achieve a homogeneoussuspension.

Step 2: An appropriate amount of solid acid was dissolved in a 4 mLglass vial with ACN.

Step 3: A volume of acid solution sufficient to obtain a desired molarratio of Example 1:acid was slowly titrated into a free base solution ona magnetic stirrer at a speed of 150 rpm. The volume of acid solutionsadd are listed in Table 5.

Step 4: The clear solution was kept stirring at room temperature withouta cap and covered by aluminum foil with pinhole for 24 hours to allowfor solvent evaporation.

Step 5: The obtained suspension was isolated by a centrifuge at a speedof 10000 rpm.

The supernatant was discarded and the resulting solid was analyzed byXRPD to determine if a new crystalline form was obtained, and then driedunder vacuum at 40° C. overnight for further characterization. Example 1and pure solid acid suspended in ACN were used as standards for Example1 and pure solid acid control, respectively.

Based on the results disclosed in Table 5 and FIG. 15 , two of thecounterions (chloride and tosylate) provided salts exhibiting goodcrystal forms. The obtained salts were recrystallized in an attempt toimprove crystallinity.

TABLE 5 Salt preparation using Method 3. Acid solution Volume Molarconcentration, of acid Acid Ratio mg/mL solution Observation XRD patternHCl 1:1 59 0.034 Clear, and then white solid Good precipitatedimmediately crystalline H₂SO₄ 1:1 61.3 0.033 Clear, and then white solidPoor precipitated immediately crystalline MsOH 1:1 49.3 0.040 Clear, andthen white solid Poor precipitated immediately crystalline TsOH 1:1 500.078 Clear, and then white solid Good precipitated after 1 hourcrystalline

Recrystallization of Method 3 Product: Four potential salts (chloride,sulfate, mesylate and tosylate) were recrystallized in several solventsin order to improve crystallinity. About 10 mg of salt candidate wasweighed into a 1.5 mL glass vial separately, and then 0.2 mL of aselected solvent (EtOH, acetone, EtOAc and THF) was added. The obtainedsuspension was agitated at 40° C. for 1 day on a thermomixer and thencentrifuged at 10000 rpm. The resulting solid was dried under vacuum at40° C. overnight for XRPD test.

XRPD results (FIG. 16 , FIG. 17 , FIG. 18 , FIG. 19 ) demonstrated thatthe crystallinity of three salts (chloride, mesylate and tosylate) wasslightly improved via this recrystallization method, employing EtOAc.

Method 3 Scale-Up: Method 3, followed by optional EtOAcrecrystallization, was repeated on a larger scale for the chloride,mesylate and tosylate salts.

Step 1: About 200 mg of Example 1 free base was dissolved in a 8 mLglass vial at 40° C. with 4 mL of ACN to achieve a homogeneoussuspension.

Step 2: An amount of solid acid was dissolved in a 4 mL glass vial withACN.

Step 3: A volume of acid solution sufficient to obtain a desired molarratio of Example 1:acid (the volume of acid solutions added are listedin Table 6) was slowly titrated into a free base solution on a magneticstirrer at 150 rpm.

Step 4: The clear solution was stirred at ambient temperature without acap and covered by aluminum foil with pinhole for 24 hours to getprecipitate.

Step 5: The obtained suspension was isolated by a centrifuge at a speedof 10000 rpm.

The supernatant was discarded and the resulting solid was analyzed byXRPD to determine if a new crystalline form was obtained, and then driedunder vacuum at 40° C. overnight for further characterization.

Based on the results listed in Table 6 and FIG. 20 , chloride, mesylateand tosylate salts were reproduced successfully. Mesylate salt (FIG. 21and Table 7) appeared to be well crystallized during salt synthesis,while both chloride and tosylate salts require recrystallization inEtOAc to improve the crystallinity.

Recrystallization of Method 3 Scale-Up Product: About 100 mg of the saltwas weighed into a 1.5 mL glass vial separately, and then 1 mL of EtOAcwas added. The obtained suspension was agitated at 40° C. for 1 day onthe thermomixer and then centrifuged at 10000 rpm. The resulting solidwas dried under vacuum at 40° C. overnight for XRPD test.

XRPD results (FIG. 22 , FIG. 23 and Table 8, Table 9) showed thecrystallinity of the chloride and tosylate salts were improved on usingthis recrystallization method. For this reason, the re-prepared chloridesalt (recrystallized in EtOAc), mesylate and tosylate (recrystallized inEtOAc) were used for further characterization.

TABLE 6 Salt preparation using Method 3 Scale-Up. Acid solution VolumeMolar concentration, of acid Acid Ratio mg/mL solution Observation XRDpattern HCl 1:1 59 0.034 Clear, and then white solid Good precipitatedimmediately crystalline H₂SO₄ 1:1 59 0.68 Clear, and then white solidGood precipitated immediately crystalline MsOH 1:1 49.3 0.80 Clear, andthen white solid Good precipitated immediately crystalline TsOH 1:1 501.57 Clear, and then white solid Good precipitated after 1 hourcrystalline

TABLE 7 XRPD peak listing of mesylate from Method 3 scale-up. 2-Thetad(Å) BG Height I % Area I % FWHM 9.176 9.6296 153 245 19.8 1880 12.70.129 10.791 8.1921 140 214 17.3 1714 11.6 0.134 13.772 6.4247 131 96978.2 14796 100.0 0.256 16.733 5.2940 126 790 63.8 8447 57.1 0.179 17.3215.1153 134 161 13.0 1879 12.7 0.196 18.385 4.8216 130 144 11.6 2268 15.30.264 18.703 4.7405 134 753 60.8 8833 59.7 0.197 19.927 4.4520 150 30624.7 4164 28.1 0.228 20.620 4.3039 165 88 7.1 1711 11.6 0.326 21.4104.1469 175 750 60.5 7402 50.0 0.165 22.054 4.0271 277 1239 100.0 1033169.8 0.140 22.337 3.9768 165 317 25.6 9076 61.3 0.480 22.647 3.9230 158289 23.3 10278 69.5 0.596 22.948 3.8723 140 326 26.3 6822 46.1 0.35124.129 3.6853 139 982 79.3 13054 88.2 0.223 32.078 2.7879 83 91 7.3 168511.4 0.310

TABLE 8 XRPD peak listing of chloride salt from Method 3 scale-up andEtOAc recrystallization. 2-Theta d(Å) BG Height I % Area I % FWHM 4.63219.0595 362 546 15.3 2967 10.9 0.091 .9.255 9.5479 153 1332 37.2 580221.2 0.073 10.951 8.0728 142 630 17.6 3095 11.3 0.082 12.584 7.0284 1491783 49.9 9975 36.5 0.094 13.246 6.6788 151 894 25.0 4362 16.0 0.08213.831 6.3976 147 2306 64.5 14227 52.1 0.103 16.497 5.3690 118 974 27.26806 24.9 0.117 18.978 4.6723 117 432 12.1 3224 11.8 0.125 20.815 4.2639126 556 15.5 4981 18.2 0.150 21.996 4.0376 140 350 9.8 2854 10.4 0.13722.392 3.9671 135 522 14.6 4813 17.6 0.155 22.693 3.9152 139 3576 100.027325 100.0 0.128 24.210 3.6732 133 376 10.5 3024 11.1 0.135 24.9773.5621 123 1070 29.9 8011 29.3 0.126 33.438 2.6776 75 175 4.9 2905 10.60.278

TABLE 9 XRPD peak listing of tosylate from Method 3 scale-up and EtOAcrecrystallization. 2-Theta d(Å) BG Height I % Area I % FWHM 4.49719.6325 446 431 20.1 2219 14.2 0.086 9.016 9.8006 190 1545 71.9 855054.7 0.093 10.255 8.6188 205 717 33.4 6223 39.8 0.146 10.536 8.3894 187502 23.4 4445 28.4 0.148 10.714 8.2506 191 714 33.2 4310 27.6 0.10111.104 7.9615 215 1242 57.8 6476 41.4 0.087 11.682 7.5690 183 362 16.91951 12.5 0.090 13.576 6.5171 162 643 29.9 3931 25.2 0.103 14.262 6.2050156 894 41.6 5475 35.0 0.103 17.066 5.1914 172 199 9.3 2097 13.4 0.17717.324 5.1146 175 1290 60.1 8180 52.4 0.106 17.640 5.0238 182 2148 100.013354 85.5 0.104 18.454 4.8040 194 364 16.9 2574 16.5 0.119 18.8804.6964 193 537 25.0 9411 60.2 0.294 19.039 4.6575 202 963 44.8 9759 62.50.170 19.179 4.6239 201 1030 48.0 13207 84.5 0.215 19.794 4.4815 222 71633.3 4549 29.1 0.107 20.069 4.4209 252 683 31.8 4899 31.4 0.120 20.4024.3493 248 799 37.2 4594 29.4 0.096 20.782 4.2708 284 1471 68.5 15624100.0 0.178 21.386 4.1514 267 891 41.5 10144 64.9 0.191 21.840 4.0661230 956 44.5 5516 35.3 0.097 22.352 3.9742 198 641 29.8 4776 30.6 0.12522.629 3.9261 188 607 28.3 7111 45.5 0.196 23.358 3.8053 168 338 15.72201 14.1 0.109 24.344 3.6532 158 282 13.1 2564 16.4 0.152 25.059 3.5506154 271 12.6 2195 14.0 0.136 26.004 3.4237 143 330 15.4 3005 19.2 0.15326.323 3.3829 140 288 13.4 2681 17.2 0.156 27.186 3.2775 132 129 6.02232 14.3 0.290 27.405 3.2518 130 126 5.9 2235 14.3 0.297 28.211 3.1606136 706 32.9 6812 43.6 0.162

Provided herein salts of Example 1 characterized by having XRPD peaks asdisclosed above. In certain embodiments, salts are characterized byhaving at least one, at least three, or at least five of the XRPD peaksas disclosed above. In certain embodiments, salts are characterized byhaving between five and ten of the XRPD peaks as disclosed above. Suchpeaks may be referred to by their 2 theta shift.

Accordingly, provided herein is the mesylate salt of Example 1 (i.e.,wherein R⁻ is CH₃SO₃ ⁻). In certain embodiments, the mesylate salt ischaracterized by having one or more x-ray powder diffraction peakschosen from about 9.2, about 10.8, about 13.8, about 16.7, about 17.3,about 18.4, about 18.7, about 19.9, about 20.6, about 21.4, about 22.1,about 22.3, about 22.6, about 22.9, about 24.1, and about 32.1 degrees2-theta. In certain embodiments, the mesylate salt is characterized byhaving two, three, four, five, or more of the peaks. In certainembodiments, the mesylate salt is characterized by having three or moreof the peaks. In certain embodiments, the mesylate salt is characterizedby having five or more of the peaks.

Accordingly, provided herein is the chloride salt of Example 1 (i.e.,wherein R⁻ is Cl⁻). In certain embodiments, the chloride salt ischaracterized by having one or more x-ray powder diffraction peakschosen from about 4.6, about 9.26, about 11.0, about 12.6, about 13.2,about 13.8, about 16.5, about 19.0, about 20.8, about 22.0, about 22.4,about 22.7, about 24.2, about 25.0, and about 33.4 degrees 2-theta. Incertain embodiments, the chloride salt is characterized by having two,three, four, five, or more of the peaks. In certain embodiments, thechloride salt is characterized by having three or more of the peaks. Incertain embodiments, the chloride salt is characterized by having fiveor more of the peaks.

Accordingly, provided herein is the tosylate salt of Example 1 (i.e.,wherein R⁻ is 4-MePhSO₃ ⁻. In certain embodiments, the chloride salt ischaracterized by having one or more x-ray powder diffraction peakschosen from about 4.5, about 9.0, about 10.3, about 10.5, about 10.7,about 11.1, about 11.7, about 13.6, about 14.3, about 17.1, about 17.3,about 17.6, about 18.5, about 18.9, about 19.0, about 19.2, about 19.8,about 20.1, about 20.4, about 20.8, about 21.4, about 21.8, about 22.4,about 22.6, about 23.4, about 24.3, about 25.1, about 26.0, about 26.3,about 27.2, about 27.4, and about 28.2 degrees 2-theta. In certainembodiments, the tosylate salt is characterized by having two, three,four, five, or more of the peaks. In certain embodiments, the tosylatesalt is characterized by having three or more of the peaks. In certainembodiments, the tosylate salt is characterized by having five or moreof the peaks.

Also provided are salts of Example, characterized by having a X-raypowder diffraction pattern as shown in the relevant Figures.

Also provided herein is the sulfate salt of Example 1 (i.e., wherein R⁻is HSO₄ ⁻.

¹HNMR results (FIG. 24 , FIG. 25 , FIG. 26 ) of the chloride, mesylate,and tosylate salts showed that the mono mesylate and mono tosylate saltswere formed, with the calculated molar ratio (API:acid) confirmed as1:1. PLM of the chloride, mesylate, and tosylate salts are depicted inFIG. 27 , FIG. 28 , and FIG. 29 , respectively. Overlays of the DSC andTGA for the chloride, mesylate, and tosylate salts are depicted in FIG.30 , FIG. 31 , and FIG. 32 , respectively.

¹H NMR of chloride salt (400 MHz, methanol-d₄) δ ppm 2.03-2.27 (m, 2H),2.74 (s, 3H), 2.84-2.96 (m, 5H), 3.08-3.21 (m, 2H), 3.25-3.31 (m, 2H),4.25-4.30 (m, 2H), 4.70-5.13 (m, 4H), 7.32 (d, J=2.51 Hz, 1H), 7.35 (d,J=2.51 Hz, 1H), 7.65 (d, J=9.29 Hz, 1H), 8.33-8.39 (m, 2H).

¹H NMR of mesylate salt (400 MHz, methanol-d₄) δ ppm 2.06-2.24 (m, 2H),2.72 (s, 3H), 2.74 (s, 3H), 2.87-2.96 (m, 5H), 3.09-3.23 (m, 2H),3.26-3.31 (m, 2H), 4.28 (s, 2H), 4.73-5.13 (m, 4H), 7.33 (dd, J=14.93,2.38 Hz, 2H), 7.65 (d, J=9.29 Hz, 1H), 8.33-8.38 (m, 2H).

¹H NMR of tosylate salt (400 MHz, methanol-d₄) δ ppm 2.04-2.27 (m, 2H),2.38 (s, 3H), 2.74 (s, 3H), 2.83-2.96 (m, 5H), 3.10-3.22 (m, 2H),3.25-3.31 (m, 2H), 4.26-4.31 (m, 2H, integrates low due to slow exchangewith solvent), 4.70-5.10 (m, 4H), 7.24 (d, J=7.78 Hz, 2H), 7.31 (d,J=2.01 Hz, 1H), 7.35 (d, J=2.26 Hz, 1H), 7.68-7.74 (m, 3H), 8.35-8.41(m, 2H).

Provided herein are salts characterized by having nuclear magneticresonance NMR peaks as disclosed above. In certain embodiments, saltsare characterized by having at least one, at least three, or at leastfive of the peaks as disclosed above. In certain embodiments, salts arecharacterized by having between five and ten of the peaks as disclosedabove. Such peaks may be referred to by their shift in parts permillion.

Provided herein is a chloride salt of Example 1 characterized by havingone or more ¹H nuclear magnetic resonance (NMR) chemical shifts at about2.0-about 2.3, about 2.7, about 2.8-about 3.0, about 3.1-about 3.2,about 3.3, about 4.3, about 4.7-about 5.1, about 7.3, about 7.4, about7.7, or about 8.3-about 8.4 parts per million. In certain embodiments,the chloride salt of Example 1 is characterized by having three or moreof the shifts. In certain embodiments, the chloride salt of Example 1 ischaracterized by having five or more of the shifts.

Provided herein is a mesylate salt of Example 1 characterized by havingone or more ¹H nuclear magnetic resonance (NMR) chemical shifts at about2.1-about 2.2, about 2.7, about 2.9-about 3.0, about 3.1-about 3.2,about 3.3, about 4.3, about 4.7-about 5.1, about 7.3, about 7.7, orabout 8.3-about 8.4 parts per million. In certain embodiments, themesylate salt of Example 1 is characterized by having three or more ofthe shifts. In certain embodiments, the mesylate salt of Example 1 ischaracterized by having five or more of the shifts.

Provided herein is a tosylate salt of Example 1 characterized by havingone or more ¹H nuclear magnetic resonance (NMR) chemical shifts at about2.0-about 2.3, about 2.4, about 2.7, about 2.8-about 3.0, about3.1-about 3.2, about 3.3, about 4.3, about 4.7-about 5.1, about 7.2,about 7.3, about 7.4, about 7.7, or about 8.4 parts per million. Incertain embodiments, the tosylate salt of Example 1 is characterized byhaving three or more of the shifts. In certain embodiments, the tosylatesalt of Example 1 is characterized by having five or more of the shifts.

Also provided are salt of Example 1 characterized by having at least oneof the XRPD peaks/shifts and at least one of the NMR peaks/shiftsdisclosed above, wherein peaks/shifts are both pertinent to theparticular salt. In certain embodiments, a salt is characterized byhaving at least two, three, four, or five of the XRPD peaks/shifts andat least three of the NMR peaks/shifts disclosed above. In certainembodiments, a salt is characterized by having at least three of theXRPD peaks/shifts and at least three of the NMR peaks/shifts disclosedabove. In certain embodiments, a salt is characterized by having atleast five of the XRPD peaks/shifts and at least five of the NMRpeaks/shifts disclosed above.

As per the PLM results (FIG. 31-33 ), 3 salt candidates displayedbirefringence phenomenon, which agreed well with previous XRPD results.The mesylate showed rod like shape, while both the chloride and tosylatesalts were irregular block like.

Thermal properties using DSC and TGA (FIG. 34-36 ) showed that for theHCl salt, a broad endothermic peak with onset temperature at 169° C.shown in FIG. 33 might be the melting of salt, and −4% weight loss from30° C. to 150° C. should be the evaporation of residual solvent. For themesylate, an endothermic peak with onset temperature at 180° C. shown inFIG. 34 might be the melting of the salt, and negligible weight lossindicates an anhydrous form. For the tosylate, an endothermic peak withonset temperature at 185° C. shown in FIG. 35 might be the melting ofthe salt, and negligible weight loss indicates an anhydrous form.

Provided herein are tosylate and mesylate salts of Example 1characterized by having XRPD peaks as disclosed above. In certainembodiments, a tosylate or mesylate salt of Example 1 is characterizedby having at least five of the XRPD peaks as disclosed in the relevanttable(s) above. In certain embodiments, a tosylate or mesylate salt ofExample 1 is characterized by having between five and ten of the XRPDpeaks as disclosed above. Such peaks may be referred to by their 2 thetashift.

In general, provided herein are salts and polymorphs of Example 1characterized by having spectral peaks (XRPD, NMR, IR, etc.) asdisclosed above and in the Figure(s) below. In certain embodiments, asalt or polymorph of Example 1 is characterized by having at least fiveof the spectral peaks as disclosed in the relevant table(s) above orFigure(s) below. In certain embodiments, a salt or polymorph of Example1 is characterized by having between five and ten of the spectral peaksas disclosed above or below. Such peaks may be referred to by their 2theta shifts (in the case of XRPD); by their chemical shift in ppm,and/or intensity, and/or grouping (in the case of NMR), and absorbanceand/or frequency (in the case of IR).

Biological Activity Assays

The following are assays that may be used to evaluate the biologicalefficacy of compounds and salts thereof disclosed herein.

GLS1 Enzymatic Activity Assay The inhibition of purified recombinanthuman GAC by varying concentrations of inhibitors is assessed via adual-coupled enzymatic assay. The glutamate produced by the glutaminasereaction is used by glutamate oxidase to produce α-ketoglutarate,ammonia, and hydrogen peroxide, with this hydrogen peroxide subsequentlybeing used by horseradish peroxidase to produce resorufin in thepresence of Amplex UltraRed. The assay buffer consisted of 50 mM Hepes(pH 7.4), 0.25 mM EDTA and 0.1 mM Triton X-100. GAC was incubated withpotassium phosphate (10 minutes at room temperature) prior to incubationwith inhibitor (10 minutes at room temperature). The final reactionconditions were as follows: 2 nM GAC, 50 mM potassium phosphate, 100mU/mL glutamate oxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mLhorseradish peroxidase (Sigma), 75 μM Amplex UltraRed (LifeTechnologies), and 1% (v/v) DMSO. The production of resorufin wasmonitored on a Perkin Elmer Envision plate reader (excitation 530 nm,emission 590 nm) either in a kinetics or endpoint mode (at 20 minutes).IC50 values were calculated using a four-parameter logistic curve fit.

Proliferation Assay. A549 cells were routinely maintained in RPMI 1640media (Gibco catalog number 11875-093) supplemented with 10% dialyzedfetal bovine serum using a humidified incubator (37° C., 5% CO2 andambient O2). In preparation for the viability assay, cells wereinoculated into 384-well black CulturPlates (Perkin Elmer) at a densityof 1000 cells/well in a volume of 40 uL. Following a 24-hour incubationat 37° C., 5% CO2 and ambient O2, cells were treated with compound (10uL) in a final DMSO concentration of 0.5% (v/v). The microplates werethen incubated for 72 hours (37° C., 5% CO2 and ambient O2). Cell TiterFluor (Promega) was subsequently added (10 uL of 6× reagent) and mixedfor 15 minutes at room temperature. The plates were then incubated for30 minutes (37° C., 5% CO2 and ambient O2) and fluorescence wassubsequently read on the Perkin Elmer Envision plate reader. IC50 valueswere calculated using a four-parameter logistic curve fit.

Example 1 had an IC50 against GLS1 and an EC₅₀ against A549 cellproliferation of <100 nM. Salts, solvates, and polymorphs of Example 1(e.g., Examples 2-11) are expected to have similar activity.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present disclosure. However, thedisclosure described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the disclosure. Anyequivalent embodiments are intended to be within the scope of thisdisclosure. Indeed, various modifications of the disclosure in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description, which do not departfrom the spirit or scope of the present inventive discovery. Suchmodifications are also intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A method of treating cancer wherein the cancer isdependent on glutamine metabolism in a subject in need thereofcomprising administering to the subject a salt or a polymorph of thecompound

wherein the salt is of structural Formula I:

R⁻ is chosen from Cl⁻, Br⁻, I⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻, CH₃SO₃ ⁻, PhSO₃⁻, 4-MePhSO₃ ⁻, and naphthaleneSO₃ ⁻; n is an integer from 1 to 2; andthe polymorph is the Polymorph D having one or more x-ray powderdiffraction peaks chosen from about 4.0, about 8.0, about 11.6, about11.9, about 14.9, about 15.9, about 17.6, about 19.9, about 20.2, about22.4, about 23.7, and about 23.9 degrees 2-theta.
 2. The method asrecited in claim 1, comprising the step of administering to the subjecta salt of the compound

wherein the salt is of structural Formula I:

R⁻ is chosen from Cl⁻, Br⁻, I⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻, CH₃SO₃ ⁻, PhSO₃⁻, 4-MePhSO₃ ⁻, and naphthaleneSO₃ ⁻; and n is an integer from 1 to 2.3. The method as recited in claim 1, wherein n is
 1. 4. The method asrecited in claim 2, wherein R⁻ is chosen from Cl⁻, HSO₄ ⁻, CH₃SO₃ ⁻, and4-MePhSO₃ ⁻.
 5. The method as recited in claim 4, wherein R⁻ is CH₃SO₃⁻.
 6. The method as recited in claim 5, wherein the mesylate salt ischaracterized by having one or more x-ray powder diffraction peakschosen from about 9.2, about 10.8, about 13.8, about 16.7, about 17.3,about 18.4, about 18.7, about 19.9, about 20.6, about 21.4, about 22.1,about 22.3, about 22.6, about 22.9, about 24.1, and about 32.1 degrees2-theta.
 7. The method as recited in claim 4, wherein R⁻ is Cl⁻.
 8. Themethod as recited in claim 7, wherein the chloride salt is characterizedby having one or more x-ray powder diffraction peaks chosen from about4.6, about 9.26, about 11.0, about 12.6, about 13.2, about 13.8, about16.5, about 19.0, about 20.8, about 22.0, about 22.4, about 22.7, about24.2, about 25.0, and about 33.4 degrees 2-theta.
 9. The method asrecited in claim 4, wherein R⁻ is 4-MePhSO₃ ⁻.
 10. The method as recitedin claim 9, wherein the tosylate salt is characterized by having one ormore x-ray powder diffraction peaks chosen from about 4.5, about 9.0,about 10.3, about 10.5, about 10.7, about 11.1, about 11.7, about 13.6,about 14.3, about 17.1, about 17.3, about 17.6, about 18.5, about 18.9,about 19.0, about 19.2, about 19.8, about 20.1, about 20.4, about 20.8,about 21.4, about 21.8, about 22.4, about 22.6, about 23.4, about 24.3,about 25.1, about 26.0, about 26.3, about 27.2, about 27.4, and about28.2 degrees 2-theta.
 11. The method as recited in claim 4, wherein R⁻is HSO₄ ⁻.
 12. The method as recited in claim 1, wherein the Polymorph Dis characterized by having two, three, four, five or more x-ray powderdiffraction peaks chosen from about 4.0, about 8.0, about 11.6, about11.9, about 14.9, about 15.9, about 17.6, about 19.9, about 20.2, about22.4, about 23.7, and about 23.9 degrees 2-theta.
 13. The method asrecited in claim 1, wherein the Polymorph D is characterized by havingfive or more x-ray powder diffraction peaks chosen from about 4.0, about8.0, about 11.6, about 11.9, about 14.9, about 15.9, about 17.6, about19.9, about 20.2, about 22.4, about 23.7, and about 23.9 degrees2-theta.
 14. The method as recited in claim 1, wherein the Polymorph Dis anhydrous.
 15. The method as recited in claim 1, wherein thePolymorph D displays an endothermic peak in DSC with onset of 197° C.±1°C.
 16. The method as recited in claim 1, further comprising thesequential or co-administration of another therapeutic agent.
 17. Themethod according to claim 16, wherein the therapeutic agent ispaclitaxel.
 18. The method of claim 1, wherein the method furthercomprises administering non-chemical methods of cancer treatment. 19.The method of claim 18, wherein the method further comprisesadministering radiation therapy.
 20. The method of claim 18, wherein themethod further comprises administering surgery, thermoablation, focusedultrasound therapy, cryotherapy, or any combination thereof.
 21. Themethod of claim 1, wherein the cancer is ovarian cancer.
 22. The methodof claim 1, wherein the cancer is non-small cell lung cancer (NSCLC).23. The method of claim 1, wherein the cancer is head and neck cancer.24. The method of claim 1, wherein the cancer is endometrial cancer. 25.The method of claim 1, wherein the cancer is melanoma.
 26. The method ofclaim 1, wherein the cancer is leukemia.
 27. The method of claim 1,wherein the cancer is renal cell cancer.
 28. The method of claim 1,wherein the cancer is breast cancer.
 29. The method of claim 1, whereinthe cancer is myeloma.
 30. The method of claim 1, wherein the cancer iscolorectal cancer.